Pyrrole Inhibitors of S-Nitrosoglutathione Reductase

ABSTRACT

The present invention is directed to inhibitors of S-nitrosoglutathione reductase (GSNOR), pharmaceutical compositions comprising such GSNOR inhibitors, and methods of making and using the same.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/057,175, filed Feb. 2, 2011, which application is a 35 U.S.C. §371national phase application of PCT/US2009/053929, filed Aug. 14, 2009 (WO2010/019909),” which claims priority to U.S. provisional applicationSer. No. 61/089,313, filed Aug. 15, 2008, each of which is incorporatedherein by reference in its entirety.

FIELD OF THE INVENTION

The present invention is directed to novel pyrrole inhibitors ofS-nitrosoglutathione reductase, pharmaceutical compositions comprisingsuch inhibitors, and methods of making and using the same.

BACKGROUND OF THE INVENTION

The chemical compound nitric oxide is a gas with chemical formula NO. NOis one of the few gaseous signaling molecules known in biologicalsystems, and plays an important role in controlling various biologicalevents. For example, the endothelium uses NO to signal surroundingsmooth muscle in the walls of arterioles to relax, resulting invasodilation and increased blood flow to hypoxic tissues. NO is alsoinvolved in regulating smooth muscle proliferation, platelet function,neurotransmission, and plays a role in host defense. Although nitricoxide is highly reactive and has a lifetime of a few seconds, it canboth diffuse freely across membranes and bind to many molecular targets.These attributes make NO an ideal signaling molecule capable ofcontrolling biological events between adjacent cells and within cells.

NO is a free radical gas, which makes it reactive and unstable, thus NOis short lived in vivo, having a half life of 3-5 seconds underphysiologic conditions. In the presence of oxygen, NO can combine withthiols to generate a biologically important class of stable NO adductscalled S-nitrosothiols (SNO's). This stable pool of NO has beenpostulated to act as a source of bioactive NO and as such appears to becritically important in health and disease, given the centrality of NOin cellular homeostasis (Stamler et al., Proc. Natl. Acad. Sci. USA,89:7674-7677 (1992)). Protein SNO's play broad roles in cardiovascular,respiratory, metabolic, gastrointestinal, immune and central nervoussystem function (Foster et al., 2003, Trends in Molecular MedicineVolume 9, Issue 4, April 2003, pages 160-168). One of the most studiedSNO's in biological systems is S-nitrosoglutathione (GSNO) (Gaston etal., Proc. Natl. Acad. Sci. USA 90:10957-10961 (1993)), an emerging keyregulator in NO signaling since it is an efficient trans-nitrosatingagent and appears to maintain an equilibrium with other S-nitrosatedproteins (Liu et al., 2001) within cells. Given this pivotal position inthe NO-SNO continuum, GSNO provides a therapeutically promising targetto consider when NO modulation is pharmacologically warranted.

In light of this understanding of GSNO as a key regulator of NOhomeostasis and cellular SNO levels, studies have focused on examiningendogenous production of GSNO and SNO proteins, which occurs downstreamfrom the production of the NO radical by the nitric oxide synthetase(NOS) enzymes. More recently there has been an increasing understandingof enzymatic catabolism of GSNO which has an important role in governingavailable concentrations of GSNO and consequently available NO andSNO's.

Central to this understanding of GSNO catabolism, researchers haverecently identified a highly conserved S-nitrosoglutathione reductase(GSNOR) (Jensen et al., Biochem J., 331:659-668 (1998); Liu et al.,Nature, 410:490-494 (2001)). GSNOR is also known asglutathione-dependent formaldehyde dehydrogenase (GS-FDH), alcoholdehydrogenase 3 (ADH-3) (Uotila and Koivusalo, Coenzymes and Cofactors.,D. Dolphin, ed. pp. 517-551 (New York, John Wiley & Sons, 1989)), andalcohol dehydrogenase 5 (ADH-5). Importantly GSNOR shows greateractivity toward GSNO than other substrates (Jensen et al., 1998; Liu etal., 2001) and appears to mediate important protein and peptidedenitrosating activity in bacteria, plants, and animals. GSNOR appearsto be the major GSNO-metabolizing enzyme in eukaryotes (Liu et al.,2001). Thus, GSNO can accumulate in biological compartments where GSNORactivity is low or absent (e.g. airway lining fluid) (Gaston et al.,1993).

Yeast deficient in GSNOR accumulate S-nitrosylated proteins which arenot substrates of the enzyme, which is strongly suggestive that GSNOexists in equilibrium with SNO-proteins (Liu et al., 2001). Preciseenzymatic control over ambient levels of GSNO and thus SNO-proteinsraises the possibility that GSNO/GSNOR may play roles across a host ofphysiological and pathological functions including protection againstnitrosative stress wherein NO is produced in excess of physiologicneeds. Indeed, GSNO specifically has been implicated in physiologicprocesses ranging from the drive to breathe (Lipton et al., Nature,413:171-174 (2001)) to regulation of the cystic fibrosis transmembraneregulator (Zaman et al., Biochem Biophys Res Commun, 284:65-70 (2001),to regulation of vascular tone, thrombosis and platelet function (deBelder et al., Cardiovasc Res. 1994 May; 28(5):691-4. (1994); Z.Kaposzta, A et al., Circulation; 106(24): 3057-3062, 2002) as well ashost defense (de Jesus-Berrios et al., Curr. Biol., 13:1963-1968(2003)). Other studies have found that GSNOR protects yeast cellsagainst nitrosative stress both in vitro (Liu et al., 2001) and in vivo(de Jesus-Berrios et al., 2003).

Collectively data suggest GSNOR as a primary physiological ligand forthe enzyme S-nitrosoglutathione reductase (GSNOR), which catabolizesGSNO and consequently reduces available SNO's and NO in biologicalsystems (Liu et al., 2001), (Liu et al., Cell, (2004), 116(4), 617-628),and (Que et al., Science, 2005, 308, (5728):1618-1621). As such, thisenzyme plays a central role in regulating local and systemic bioactiveNO. Since perturbations in NO bioavailability has been linked to thepathogenesis of numerous disease states, including hypertension,atherosclerosis, thrombosis, asthma, gastrointestinal disorders,inflammation and cancer, agents that regulate GSNOR activity arecandidate therapeutic agents for treating diseases associated withnitric oxide imbalance.

Currently, there is a great need in the art for diagnostics,prophylaxis, ameliorations, and treatments for medical conditionsrelating to increased NO synthesis and/or increased NO bioactivity. Inaddition, there is a significant need for novel compounds, compositionsand methods for preventing, ameliorating, or reversing otherNO-associated disorders. The present invention satisfies these needs.

SUMMARY OF THE INVENTION

The present invention provides novel pyrrole compounds useful asS-nitrosoglutathione reductase (“GSNOR”) inhibitors. The inventionencompasses pharmaceutically acceptable salts, prodrugs, and metabolitesof the described GSNOR inhibitors. Also encompassed by the invention arepharmaceutical compositions comprising at least one GSNOR inhibitor andat least one pharmaceutically acceptable carrier.

The compositions of the present invention can be prepared in anysuitable pharmaceutically acceptable dosage form.

The present invention provides a method for inhibitingS-nitrosoglutathione reductase in a subject in need thereof. Such amethod comprises administering a therapeutically effective amount of apharmaceutical composition comprising at least one GSNOR inhibitor or apharmaceutically acceptable salt thereof, a prodrug or metabolitethereof, in combination with at least one pharmaceutically acceptablecarrier. The GSNOR inhibitor can be a novel compound according to theinvention, or it can be a known compound which previously was not knownto be an inhibitor of GSNOR.

The present invention also provides a method of treating a disorderameliorated by NO donor therapy in a subject in need thereof. Such amethod comprises administering a therapeutically effective amount of apharmaceutical composition comprising at least one GSNOR inhibitor or apharmaceutically acceptable salt thereof, a prodrug, or metabolitethereof, in combination with at least one pharmaceutically acceptablecarrier. The GSNOR inhibitor can be a novel compound according to theinvention, or it can be a known compound which previously was not knownto be an inhibitor of GSNOR.

The present invention also provides a method of treating a cellproliferative disorder in a subject in need thereof. Such a methodcomprises administering a therapeutically effective amount of apharmaceutical composition comprising at least one GSNOR inhibitor or apharmaceutically acceptable salt thereof, a prodrug, or metabolitethereof, in combination with at least one pharmaceutically acceptablecarrier. The GSNOR inhibitor can be a novel compound according to theinvention, or it can be a known compound which previously was not knownto be an inhibitor of GSNOR.

The methods of the invention encompass administration with one or moresecondary active agents. Such administration can be sequential or in acombination composition.

Although methods and materials similar or equivalent to those describedherein can be used in the practice or testing of the present invention,suitable methods and materials are described below. All publiclyavailable publications, patent applications, patents, and otherreferences mentioned herein are incorporated by reference in theirentirety. In the case of conflict, the present specification, includingdefinitions, will control.

Both the foregoing summary and the following detailed description areexemplary and explanatory and are intended to provide further details ofthe compositions and methods as claimed. Other objects, advantages, andnovel features will be readily apparent to those skilled in the art fromthe following detailed description.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS A. Overview of theInvention

Until recently, S-nitrosoglutathione reductase (GSNOR) was known tooxidize the formaldehyde glutathione adduct, S-hydroxymethylglutathione.GSNOR has since been identified in a variety of bacteria, yeasts, plantsand animals and is well conserved. The proteins from E. coli, S.cerevisiae and mouse macrophages share over 60% amino acid sequenceidentity. GSNOR activity (i.e., decomposition of S-nitrosoglutathionewhen NADH is present as a required cofactor) has been detected in E.coli, in mouse macrophages, in mouse endothelial cells, in mouse smoothmuscle cells, in yeasts, and in human HeLa, epithelial and monocytecells. Human GSNOR nucleotide and amino acid sequence information can beobtained from the National Center for Biotechnology Information (NCBI)databases under Accession Nos. M29872, NM_(—)000671. Mouse GSNORnucleotide and amino acid sequence information can be obtained from NCBIdatabases under Accession Nos. NM_(—)007410. In the nucleotide sequence,the start site and stop site are underlined. CDS designates codingsequence. SNP designates single nucleotide polymorphism. Other relatedGSNOR nucleotide and amino acid sequences, including those of otherspecies, can be found in U.S. Patent Application 2005/0014697.

In accord with the present invention, GSNOR has been shown to functionin vivo and in vitro to metabolize S-nitrosoglutathione (GSNO) andprotein S-nitrosothiols (SNOs) to modulate NO bioactivity, bycontrolling the intracellular levels of low mass NO donor compounds andpreventing protein nitrosylation from reaching toxic levels.

Based on this, it follows that inhibition of this enzyme potentiatesbioactivity in all diseases in which NO donor therapy is indicated,inhibits the proliferation of pathologically proliferating cells, andincreases NO bioactivity in diseases where this is beneficial.

The present invention provides pharmaceutical agents that are potentinhibitors of GSNOR. In particular, provided are substituted pyrroleanalogs that are inhibitors of GSNOR having the structures depictedbelow (Formula I), or a pharmaceutically acceptable salt, stereoisomer,or prodrug thereof.

Tri-substituted pyrrole analogs are potent inhibitors of GSNOR. As usedin this context, the term “analog” refers to a compound having similarchemical structure and function as compounds of Formula I that retainsthe pyrrole ring.

Some pyrrole analogs of the invention can also exist in various isomericforms, including configurational, geometric and conformational isomers,as well as existing in various tautomeric forms, particularly those thatdiffer in the point of attachment of a hydrogen atom. As used herein,the term “isomer” is intended to encompass all isomeric forms of acompound including tautomeric forms of the compound.

Illustrative compounds having asymmetric centers can exist in differentenantiomeric and diastereomeric forms. A compound can exist in the formof an optical isomer or a diastereomer. Accordingly, the inventionencompasses compounds in the forms of their optical isomers,diastereomers and mixtures thereof, including racemic mixtures.

It should be noted that if there is a discrepancy between a depictedstructure and a name given to that structure, the depicted structurecontrols. In addition, if the stereochemistry of a structure or aportion of a structure is not indicated with, for example, bold, wedged,or dashed lines, the structure or portion of the structure is to beinterpreted as encompassing all stereoisomers of the described compound.

In accordance with the invention, the levels of the S-nitrosoglutathionereductase in the biological sample can be determined by the methodsdescribed in U.S. Patent Application Publication No. 2005/0014697. Theterm “biological sample” includes, but is not limited to, samples ofblood (e.g., serum, plasma, or whole blood), urine, saliva, sweat,breast milk, vaginal secretions, semen, hair follicles, skin, teeth,bones, nails, or other secretions, body fluids, tissues, or cells.

B. Definitions

As used herein, “about” will be understood by persons of ordinary skillin the art and will vary to some extent on the context in which it isused. If there are uses of the term which are not clear to persons ofordinary skill in the art given the context in which it is used, “about”will mean up to plus or minus 10% of the particular term.

The term “acyl” includes compounds and moieties that contain the acetylradical (CH₃CO—) or a carbonyl group to which a straight or branchedchain lower alkyl residue is attached.

The term “alkyl” as used herein refers to a straight or branched chain,saturated hydrocarbon having the indicated number of carbon atoms. Forexample, (C₁-C₆) alkyl is meant to include, but is not limited tomethyl, ethyl, propyl, isopropyl, butyl, sec-butyl, tert-butyl, pentyl,isopentyl, neopentyl, hexyl, isohexyl, and neohexyl. An alkyl group canbe unsubstituted or optionally substituted with one or more substituentsas described herein.

The term “alkenyl” as used herein refers to a straight or branched chainunsaturated hydrocarbon having the indicated number of carbon atoms andat least one double bond. Examples of a (C₂-C₈) alkenyl group include,but are not limited to, ethylene, propylene, 1-butylene, 2-butylene,isobutylene, sec-butylene, 1-pentene, 2-pentene, isopentene, 1-hexene,2-hexene, 3-hexene, isohexene, 1-heptene, 2-heptene, 3-heptene,isoheptene, 1-octene, 2-octene, 3-octene, 4-octene, and isooctene. Analkenyl group can be unsubstituted or optionally substituted with one ormore substituents as described herein.

The term “alkynyl” as used herein refers to a straight or branched chainunsaturated hydrocarbon having the indicated number of carbon atoms andat least one triple bond. Examples of a (C₂-C₈) alkynyl group include,but are not limited to, acetylene, propyne, 1-butyne, 2-butyne,1-pentyne, 2-pentyne, 1-hexyne, 2-hexyne, 3-hexyne, 1-heptyne,2-heptyne, 3-heptyne, 1-octyne, 2-octyne, 3-octyne and 4-octyne. Analkynyl group can be unsubstituted or optionally substituted with one ormore substituents as described herein.

The term “alkoxy” as used herein refers to an —O-alkyl group having theindicated number of carbon atoms. For example, a (C₁-C₆) alkoxy groupincludes —O-methyl, —O-ethyl, —O-propyl, —O-isopropyl, —O-butyl,—O-sec-butyl, —O-tert-butyl, —O-pentyl, —O-isopentyl, —O-neopentyl,—O-hexyl, —O-isohexyl, and —O-neohexyl.

The term “aminoalkyl” as used herein, refers to an alkyl group(typically one to six carbon atoms) wherein one or more of the C₁-C₆alkyl group's hydrogen atoms is replaced with an amine of formula—N(R^(c))₂, wherein each occurrence of R^(c) is independently —H or(C₁-C₆) alkyl. Examples of aminoalkyl groups include, but are notlimited to, —CH₂NH₂, —CH₂CH₂NH₂, —CH₂CH₂CH₂NH₂, —CH₂CH₂CH₂CH₂NH₂,—CH₂CH₂CH₂CH₂CH₂NH₂, —CH₂CH₂CH₂CH₂CH₂CH₂NH₂, —CH₂CH₂CH₂N(CH₃)₂,t-butylaminomethyl, isopropylaminomethyl and the like.

The term “aryl” as used herein refers to a 5- to 14-membered monocyclic,bicyclic or tricyclic aromatic ring system. Examples of an aryl groupinclude phenyl and naphthyl. An aryl group can be unsubstituted oroptionally substituted with one or more substituents as described hereinbelow. Examples of aryl groups include phenyl or aryl heterocycles suchas, pyrrole, furan, thiophene, thiazole, isothiazole, imidazole,triazole, tetrazole, pyrazole, oxazole, isoxazole, pyridine, pyrazine,pyridazine, and pyrimidine, and the like.

As used herein, the term “bioactivity” indicates an effect on one ormore cellular or extracellular process (e.g., via binding, signaling,etc.) which can impact physiological or pathophysiological processes.

The term “carbonyl” or “carboxy” or “carboxyl” includes compounds andmoieties which contain a carbon connected with a double bond to anoxygen atom. Examples of moieties containing a carbonyl include, but arenot limited to, aldehydes, ketones, carboxylic acids, amides, esters,anhydrides, etc.

The term “C_(m)-C_(n)” means “m” number of carbon atoms to “n” number ofcarbon atoms. For example, the term “C₁-C₆” means one to six carbonatoms (C₁, C₂, C₃, C₄, C₅ or C₆). The term “C₂-C₆” includes two to sixcarbon atoms (C₂, C₃, C₄, C₅ or C₆). The term “C₃-C₆” includes three tosix carbon atoms (C₃, C₄, C₅ or C₆).

The term “cycloalkyl” as used herein refers to a 3- to 14-memberedsaturated or unsaturated non-aromatic monocyclic, bicyclic or tricyclichydrocarbon ring system. Included in this class are cycloalkyl groupswhich are fused to a benzene ring. Representative cycloalkyl groupsinclude, but are not limited to, cyclopropyl, cyclobutyl, cyclobutenyl,cyclopentyl, cyclopentenyl, cyclopentadienyl, cyclohexyl, cyclohexenyl,1,3-cyclohexadienyl, cycloheptyl, cycloheptenyl, 1,3-cycloheptadienyl,1,4-cycloheptadienyl, -1,3,5-cycloheptatrienyl, cyclooctyl,cyclooctenyl, 1,3-cyclooctadienyl, 1,4-cyclooctadienyl,-1,3,5-cyclooctatrienyl, decahydronaphthalene, octahydronaphthalene,hexahydronaphthalene, octahydroindene, hexahydroindene, tetrahydroinden,decahydrobenzocycloheptene, octahydrobenzocycloheptene,hexahydrobenzocycloheptene, tetrahydrobenzocyclopheptene,dodecahydroheptalene, decahydroheptalene, octahydroheptalene,hexahydroheptalene, and tetrahydroheptalene,(1s,3s)-bicyclo[1.1.0]butane, bicyclo[1.1.1]pentane,bicyclo[2.1.1]hexane, Bicyclo[2.2.1]heptane, bicyclo[2.2.2]octane,bicyclo[3.1.1]heptane, bicyclo[3.2.1]octane, bicyclo[3.3.1]nonane,bicyclo[3.3.2]decane, bicyclo[3.3.]undecane, bicyclo[4.2.2]decane,bicyclo[4.3.1]decane. A cycloalkyl group can be unsubstituted oroptionally substituted with one or more substituents as described hereinbelow.

The term “halogen” includes fluorine, bromine, chlorine, iodine, etc.

The term “haloalkyl” as used herein, refers to a C₁-C₆ alkyl groupwherein from one or more of the C₁-C₆ alkyl group's hydrogen atom isreplaced with a halogen atom, which can be the same or different.Examples of haloalkyl groups include, but are not limited to,trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl,pentachloroethyl, and 1,1,1-trifluoro-2-bromo-2-chloroethyl.

The term “heteroalkyl” by itself or in combination with another term,means, unless otherwise stated, a stable straight or branched chainalkyl, or combinations thereof, consisting of carbon atoms and from oneto three heteroatoms selected from the group consisting of O, N and S,and wherein the nitrogen and sulfur atoms may optionally be oxidized andthe nitrogen heteroatom may optionally be quaternized. The heteroatom(s)O, N and S can be placed at any position of the heteroalkyl group.Examples include —CH₂—CH₂—O—CH₃, —CH₂—CH₂—NH—CH₃, —CH₂—CH₂—N(CH₃)—CH₃,—CH₂—S—CH₂—CH₃, —CH₂—CH₂—S(O)—CH₃, —CH₂—CH₂—S(O)₂—CH₃, and—CH₂—CH═N—OCH₃. Up to two heteroatoms can be consecutive, such as, forexample, —CH₂—NH—OCH₃. When a prefix such as (C₂-C₈) is used to refer toa heteroalkyl group, the number of carbons (2 to 8, in this example) ismeant to include the heteroatoms as well. For example, a C₂-heteroalkylgroup is meant to include, for example, —CH₂OH (one carbon atom and oneheteroatom replacing a carbon atom) and —CH₂SH.

To further illustrate the definition of a heteroalkyl group, where theheteroatom is oxygen, a heteroalkyl group can be an oxyalkyl group. Forinstance, (C2-C5) oxyalkyl is meant to include, for example —CH₂—O—CH₃(a C₃-oxyalkyl group with two carbon atoms and one oxygen replacing acarbon atom), —CH₂CH₂CH₂CH₂OH, —OCH₂CH₂OCH₂CH₂OH, —OCH₂CH(OH)CH₂OH, andthe like.

The term “heteroaryl” as used herein refers to an aromatic heterocyclering of 5 to 14 members and having at least one heteroatom selected fromnitrogen, oxygen and sulfur, and containing at least 1 carbon atom,including monocyclic, bicyclic, and tricyclic ring systems.Representative heteroaryls are triazolyl, tetrazolyl, oxadiazolyl,pyridyl, furyl, benzofuranyl, thienyl, benzothienyl, quinolinyl,pyrrolyl, indolyl, oxazolyl, benzoxazolyl, imidazolyl, benzimidazolyl,thiazolyl, benzothiazolyl, isoxazolyl, pyrazolyl, isothiazolyl,pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, cinnolinyl,phthalazinyl, quinazolinyl, pyrimidyl, azepinyl, oxepinyl, quinoxalinyland oxazolyl. A heteroaryl group can be unsubstituted or optionallysubstituted with one or more substituents as described herein below.

As used herein, the term “heteroatom” is meant to include oxygen (O),nitrogen (N), and sulfur (S).

As used herein, the term “heterocycle” refers to 3- to 14-membered ringsystems which are either saturated, unsaturated, or aromatic, and whichcontains from 1 to 4 heteroatoms independently selected from nitrogen,oxygen and sulfur, and wherein the nitrogen and sulfur heteroatoms canbe optionally oxidized, and the nitrogen heteroatom can be optionallyquaternized, including, including monocyclic, bicyclic, and tricyclicring systems. The bicyclic and tricyclic ring systems may encompass aheterocycle or heteroaryl fused to a benzene ring. The heterocycle canbe attached via any heteroatom or carbon atom, where chemicallyacceptable. Heterocycles include heteroaryls as defined above.Representative examples of heterocycles include, but are not limited to,aziridinyl, oxiranyl, thiiranyl, triazolyl, tetrazolyl, azirinyl,diaziridinyl, diazirinyl, oxaziridinyl, azetidinyl, azetidinonyl,oxetanyl, thietanyl, piperidinyl, piperazinyl, morpholinyl, pyrrolyl,oxazinyl, thiazinyl, diazinyl, dioxanyl, triazinyl, tetrazinyl,imidazolyl, tetrazolyl, pyrrolidinyl, isoxazolyl, furanyl, furazanyl,pyridinyl, oxazolyl, benzoxazolyl, benzisoxazolyl, thiazolyl,benzthiazolyl, thienyl, pyrazolyl, triazolyl, pyrimidinyl,benzimidazolyl, isoindolyl, indazolyl, benzodiazolyl, benzotriazolyl,benzoxazolyl, benzisoxazolyl, purinyl, indolyl, isoquinolinyl,quinolinyl and quinazolinyl. A heterocycle group can be unsubstituted oroptionally substituted with one or more substituents as described hereinbelow.

The term “heterocycloalkyl” by itself or in combination with otherterms, represents, unless otherwise stated, cyclic versions of“heteroalkyl.” Additionally, a heteroatom can occupy the position atwhich the heterocycle is attached to the remainder of the molecule.Examples of heterocycloalkyl include 1-(1,2,5,6-tetrahydropyridyl),1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl,3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl,tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1-piperazinyl,2-piperazinyl, and the like.

The term “hydroxyalkyl” as used herein, refers to an alkyl group havingthe indicated number of carbon atoms wherein one or more of the hydrogenatoms in the alkyl group is replaced with an —OH group. Examples ofhydroxyalkyl groups include, but are not limited to, —CH₂OH, —CH₂CH₂OH,—CH₂CH₂CH₂OH, —CH₂CH₂CH₂CH₂OH, —CH₂CH₂CH₂CH₂CH₂OH,—CH₂CH₂CH₂CH₂CH₂CH₂OH, and branched versions thereof.

The term “hydroxy” or “hydroxyl” includes groups with an —OH or —O⁻.

As used herein and unless otherwise indicated, the term “stereoisomer”means one stereoisomer of a compound that is substantially free of otherstereoisomers of that compound. For example, a stereomerically purecompound having one chiral center will be substantially free of theopposite enantiomer of the compound. A stereomerically pure compoundhaving two chiral centers will be substantially free of otherdiastereomers of the compound. In some embodiments, a stereomericallypure compound comprises greater than about 80% by weight of onestereoisomer of the compound and less than about 20% by weight of otherstereoisomers of the compound, for example greater than about 90% byweight of one stereoisomer of the compound and less than about 10% byweight of the other stereoisomers of the compound, or greater than about95% by weight of one stereoisomer of the compound and less than about 5%by weight of the other stereoisomers of the compound, or greater thanabout 97% by weight of one stereoisomer of the compound and less thanabout 3% by weight of the other stereoisomers of the compound.

As used herein, “protein” is used synonymously with “peptide,”“polypeptide,” or “peptide fragment”. A “purified” polypeptide, protein,peptide, or peptide fragment is substantially free of cellular materialor other contaminating proteins from the cell, tissue, or cell-freesource from which the amino acid sequence is obtained, or substantiallyfree from chemical precursors or other chemicals when chemicallysynthesized.

As used herein, “modulate” is meant to refer to an increase or decreasein the levels of a peptide or a polypeptide, or to increase or decreasethe stability or activity of a peptide or a polypeptide. The term“inhibit” is meant to refer to a decrease in the levels of a peptide ora polypeptide or to decrease in the stability or activity of a peptideor a polypeptide. In preferred embodiments, the peptide which ismodulated or inhibited is S-nitrosoglutathione (GSNO) or proteinS-nitrosothiols (SNOs).

As used here, the terms “nitric oxide” and “NO” encompass unchargednitric oxide and charged nitric oxide species, particularly includingnitrosonium ion (NO⁺) and nitroxyl ion (NO⁻). The reactive form ofnitric oxide can be provided by gaseous nitric oxide. Compounds havingthe structure X—NO_(y) wherein X is a nitric oxide releasing, deliveringor transferring moiety, including any and all such compounds whichprovide nitric oxide to its intended site of action in a form active fortheir intended purpose, and Y is 1 or 2.

As utilized herein, the term “pharmaceutically acceptable” meansapproved by a regulatory agency of a federal or a state government orlisted in the U.S. Pharmacopoeia or other generally recognizedpharmacopoeia for use in animals and, more particularly, in humans. Theterm “carrier” refers to a diluent, adjuvant, excipient, or vehicle withwhich the therapeutic is administered and includes, but is not limitedto such sterile liquids as water and oils.

A “pharmaceutically acceptable salt” or “salt” of a GSNOR inhibitor is aproduct of the disclosed compound that contains an ionic bond, and istypically produced by reacting the disclosed compound with either anacid or a base, suitable for administering to a subject. Apharmaceutically acceptable salt can include, but is not limited to,acid addition salts including hydrochlorides, hydrobromides, phosphates,sulphates, hydrogen sulphates, alkylsulphonates, arylsulphonates,arylalkylsulfonates, acetates, benzoates, citrates, maleates, fumarates,succinates, lactates, and tartrates; alkali metal cations such as Li,Na, K, alkali earth metal salts such as Mg or Ca, or organic aminesalts.

A “pharmaceutical composition” is a formulation comprising the disclosedcompounds in a form suitable for administration to a subject. Apharmaceutical composition of the invention is preferably formulated tobe compatible with its intended route of administration. Examples ofroutes of administration include, but are not limited to, oral andparenteral, e.g., intravenous, intradermal, subcutaneous, inhalation,topical, transdermal, transmucosal, and rectal administration.

The term “substituted,” as used herein, means that any one or morehydrogens on the designated atom is replaced with a selection from theindicated group, provided that the designated atom's normal valency isnot exceeded, and that the substitution results in a stable compound.When a substituent is keto (i.e., ═O), then 2 hydrogens on the atom arereplaced. Ring double bonds, as used herein, are double bonds that areformed between two adjacent ring atoms (e.g., C═C, C═N, or N═N).

Substituents for the groups referred to as alkyl, heteroalkyl, alkylene,alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl andheterocycloalkenyl can be selected from a variety of groups including—OR^(d)′, ═O, ═NR^(d)′, ═N—OR^(d)′, —NR^(d)′R^(d)″, —SR^(d)′, -halo,—SiR^(d)′R^(d)″R^(d)′″, —OC(O)R^(d)′, —C(O)R^(d)′, —CO₂R^(d)′,—CONR^(d)′R^(d)″, —OC(O)NR^(d)′R^(d)″, —NR^(d)″C(O)R^(d)′,—NR^(d)′″C(O)NR^(d)′R^(d)″, —NR^(d)′″SO₂NR^(d)′R^(d)″,—NR^(d)″CO₂R^(d)′, —NHC(NH₂)═NH, —NR^(a)′C(NH₂)═NH, —NHC(NH₂)═NR^(d)′,—S(O)R^(d)′, —SO₂R^(d)′, —SO₂NR^(d)′R^(d)″, —NR^(d)″SO₂R^(d)′, —CN and—NO₂, in a number ranging from zero to three, with those groups havingzero, one or two substituents being exemplary.

R^(d)′, R^(d)″ and R^(d)′″ each independently refer to hydrogen,unsubstituted (C₁-C₈)alkyl, unsubstituted hetero(C₁-C₈) alkyl,unsubstituted aryl and aryl substituted with one to three substituentsselected from -halo, unsubstituted alkyl, unsubstituted alkoxy,unsubstituted thioalkoxy and unsubstituted aryl (C₁-C₄)alkyl. WhenR^(d)′ and R^(d)″ are attached to the same nitrogen atom, they can becombined with the nitrogen atom to form a 5-, 6- or 7-membered ring. Forexample, —NR^(d)′R^(d)″ can represent 1-pyrrolidinyl or 4-morpholinyl.

Typically, an alkyl or heteroalkyl group will have from zero to threesubstituents, with those groups having two or fewer substituents beingexemplary of the present invention. An alkyl or heteroalkyl radical canbe unsubstituted or monosubstituted. In some embodiments, an alkyl orheteroalkyl radical will be unsubstituted.

Exemplary substituents for the alkyl and heteroalkyl radicals includebut are not limited to —OR″, ═O, ═NR^(d)′, ═N—OR^(d)′, —NR^(d)′R^(d)″,—SR^(d)′, -halo, —SiR^(d)′R^(d)″R^(d)′″, —OC(O)R^(d)′, —C(O)R^(d)′,—CO₂R^(d)′, —CONR^(d)′R^(d)″, —OC(O)NR^(d)′R^(d)″, —NR^(d)″C(O)R^(d)′,—NR^(d)′″C(O)NR^(d)′R^(d)″, —NR^(d)′″SO₂NR^(d)′R^(d)″,—NR^(d)″CO₂R^(d)′, —NHC(NH₂)═NH, —NR^(a)′C(NH₂)═NH, —NHC(NH₂)═NR^(d)′,—S(O)R^(d)′, —SO₂R^(d)′, —SO₂NR^(d)′R^(d)″, —NR^(d)″SO₂R^(d)′, —CN and—NO₂, where R^(d)′, R^(d)″ and R^(d)′″ are as defined above. Typicalsubstituents can be selected from: —OR″, ═O, —NR^(d)′R^(d)″, -halo,—OC(O)R^(d)′, —CO₂R^(d)′, —C(O)NR^(d)′R^(d)″, —OC(O)NR^(d)′R^(d)″,—NR^(d)″C(O)R^(d)′, —NR^(d)″CO₂R^(d)′, —NR^(d)′″SO₂NR^(d)′R^(d)″,—SO₂R^(d)′, —SO₂NR^(d)′R^(d)″, —NR^(d)″SO₂R^(d)′-CN and —NO₂.

Similarly, substituents for the aryl and heteroaryl groups are variedand selected from: -halo, —OR^(e)′, —OC(O)R^(e)′, —NR^(e)′R^(e)″, —SR′,—R^(e)′, —CN, —NO₂, —CO₂R^(e)′, —C(O)NR^(e)′R^(e)″, —C(O)R^(e)′,—OC(O)NR^(e)′R^(e)″, —NR^(e)″C(O)R^(e)′, —NR^(e)″CO₂R^(e)′,—NR^(e)′″C(O)NR^(e)′R^(e)″, —NR^(e)′″SO₂NR^(e)′R^(e)″, —NHC(NH₂)═NH,—NR^(e)′C(NH₂)═NH, —NH—C(NH₂)═NR^(e)′, —S(O)R^(e)′, —SO₂R^(e)′,—SO₂NR^(e)′R^(e)″, —NR^(e)″SO₂R^(e)′, —N₃, —CH(Ph)₂, perfluoroalkoxy andperfluoro(C₁-C₄)alkyl, in a number ranging from zero to the total numberof open valences on the aromatic ring system.

R^(e)′, R^(e)″ and R^(e)′″ are independently selected from hydrogen,unsubstituted (C₁-C₈) alkyl, unsubstituted hetero(C₁-C₈) alkyl,unsubstituted aryl, unsubstituted heteroaryl, unsubstituted aryl(C₁-C₄)alkyl and unsubstituted aryloxy(C₁-C₄) alkyl. Typically, an aryl orheteroaryl group will have from zero to three substituents, with thosegroups having two or fewer substituents being exemplary in the presentinvention. In one embodiment of the invention, an aryl or heteroarylgroup will be unsubstituted or monosubstituted. In another embodiment,an aryl or heteroaryl group will be unsubstituted.

Two of the substituents on adjacent atoms of an aryl or heteroaryl ringin an aryl or heteroaryl group as described herein may optionally bereplaced with a substituent of the formula -T-C(O)—(CH₂)_(q)—U—, whereinT and U are independently —NH—, —O—, —CH₂— or a single bond, and q is aninteger of from 0 to 2. Alternatively, two of the substituents onadjacent atoms of the aryl or heteroaryl ring may optionally be replacedwith a substituent of the formula -J-(CH₂)_(r)—K—, wherein J and K areindependently —CH₂—, —O—, —NH—, —S—, —S(O)—, —S(O)₂—, —S(O)₂NR^(f)′— ora single bond, and r is an integer of from 1 to 3. One of the singlebonds of the new ring so formed may optionally be replaced with a doublebond. Alternatively, two of the substituents on adjacent atoms of thearyl or heteroaryl ring may optionally be replaced with a substituent ofthe formula —(CH₂)_(s)—X—(CH₂)_(t)—, where s and t are independentlyintegers of from 0 to 3, and X is —O—, —NR^(f)′—, —S—, —S(O)—, —S(O)₂—,or —S(O)₂NR^(a)′—. The substituent R^(f)′ in —NR^(f)′— and—S(O)₂NR^(f)′— is selected from hydrogen or unsubstituted (C₁-C₆) alkyl.

“Stable compound” and “stable structure” are meant to indicate acompound that is sufficiently robust to survive isolation to a usefuldegree of purity from a reaction mixture, and formulation into anefficacious therapeutic agent.

As used herein the term “therapeutically effective amount” generallymeans the amount necessary to ameliorate at least one symptom of adisorder to be prevented, reduced, or treated as described herein. Thephrase “therapeutically effective amount” as it relates to the GSNORinhibitors of the present invention shall mean the GSNOR inhibitordosage that provides the specific pharmacological response for which theGSNOR inhibitor is administered in a significant number of subjects inneed of such treatment. It is emphasized that a therapeuticallyeffective amount of a GSNOR inhibitor that is administered to aparticular subject in a particular instance will not always be effectivein treating the conditions/diseases described herein, even though suchdosage is deemed to be a therapeutically effective amount by those ofskill in the art.

C. S-Nitrosoglutathione Reductase Inhibitors

1. Inventive Compounds

In one of its aspects the present invention provides a compound having astructure shown in Formula I, or a pharmaceutically acceptable salt,stereoisomer, or prodrug thereof:

whereinR₁ is selected from the group consisting R₅ or R₆;R₁′ is R₅ when R₁ is R₆, and R₆ when R₁ is R₅;R₂ is selected from the group consisting of hydrogen, halogen, hydroxyl,C₁-C₆ alkyl, C₃-C₆ cycloalkyl, cyano, nitro, CF₃, carbamoyl, C₁-C₆alkylcarbamoyl, amino, C₁-C₆ alkylamino, C₁-C₆ dialkylamino, C₁-C₆alkoxyl, and C₃-C₆ cycloalkoxyl;R₃ is selected from the group consisting of halogen, hydroxyl,carbamoyl, substituted carbamoyl, C₁-C₆ alkylcarbamoyl, sulfamoyl, C₁-C₆alkylsulfamoyl, C₁-C₆ alkyl, C₃-C₆ cycloalkyl, cyano, nitro, amino,trifluoromethyl, carboxyl, ureido, sulfamoylamino, 2-amino-2-oxoethyl,C₁-C₆ alkylamino, C₁-C₆ dialkylamino, arylamino, heteroarylamino, C₁-C₆alkoxyl, C₃-C₆ cycloalkoxyl, aryl, substituted aryl, heteroaryl, andsubstituted heteroaryl;R₄ is selected from the group consisting of hydrogen, hydroxyl, halogen,C₁-C₆ alkyl, C₃-C₆ cycloalkyl, cyano, nitro, carbamoyl, C₁-C₆alkylcarbamoyl, sulfamoyl, C₁-C₆ alkyl sulfamoyl, amino, C₁-C₆alkylamino, C₁-C₆ dialkylamino, C₁-C₆ alkoxyl, and C₃-C₆ cycloalkoxyl;R₅ is selected from the group consisting of aryl, substituted aryl,heteroaryl, or substituted heteroaryl;R₆ is selected from the group consisting of —CR₇R₈CR₉R₁₀(CR₁₁R₁₂)_(n)R₁₃and

R₇, R₈, R₉, R₁₀, R₁₁, and R₁₂ are each independently selected fromhydrogen, hydroxyl, halogen, C₁-C₆ alkyl, C₃-C₆ cycloalkyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl and wherein R₉ andR₁₀ together form a C₃-C₆ cycloalkyl;R₁₃ is selected from the group consisting of COOH and NH₂SO₂CF₃;R₁₄ is selected from the group consisting of OH, COOH, and SO₂NH₂;R₁₅ and R₁₆ are independently selected from the group consisting ofhydrogen, hydroxyl, carboxyl, NO₂, halogen, cyano, and carbamoyl;n is 0 or 1; andX and Y are independently selected from the group consisting of C and N.

In a further aspect of the invention, suitable identities for R₅include, but are not limited to, 4-chlorophenyl, 3-chlorophenyl,4-chloro-2-methoxyphenyl, 4-bromophenyl, 3-bromophenyl, 4-fluorophenyl,3-fluorophenyl, 4-hydroxyphenyl, 4-methoxyphenyl, 3-methoxyphenyl,2-methoxyphenyl, 4-chlorothiophen-2-yl, 5-chlorothiophen-2-yl,3-bromothiophen-2-yl, 4-bromothiophen-2-yl, 5-bromothiopheny-2-yl,5-bromothiophen-3-yl, and

whereinR₁₇ is selected from the group consisting of hydrogen, methyl, chloro,fluoro, hydroxy, methoxy, ethoxy, propoxy, carbamoyl, dimethylamino,amino, formamido, and trifluoromethyl; andR₁₈ is selected from the group consisting of hydrogen, methyl, andethyl.

In a further aspect of the invention, suitable identities for R₂include, but are not limited to, C₁-C₆ alkyl, suitable identity for R₃includes, but is not limited to carbamoyl, and suitable identity for R₄includes, but is not limited to hydrogen.

In a further aspect of the invention, suitable identities for R₅include, but are not limited to, 4-methoxyphenyl,4-chloro-2-methoxyphenyl, 4-(1H-imidazol-1-yl)phenyl,4-(2-methyl-1H-imidazol-1-yl)phenyl.

In a further aspect of the invention, suitable compounds of formula Iinclude, but are not limited to:

-   3-(2-(4-carbamoyl-2-methylphenyl)-3-(4-methoxyphenyl)-1H-pyrrol-1-yl)propanoic    acid;-   3-(2-(4-carbamoyl-2-methylphenyl)-3-(4-methoxyphenyl)-1H-pyrrol-1-yl)-3-phenylpropanoic    acid;-   3-(2-(4-carbamoyl-2-methylphenyl)-3-(4-methoxyphenyl)-1H-pyrrol-1-yl)-2-methylpropanoic    acid;-   4-(2-(4-carbamoyl-2-methylphenyl)-3-(4-methoxyphenyl)-1H-pyrrol-1-yl)butanoic    acid;-   3-(3-(4-(1H-imidazol-1-yl)phenyl)-2-(4-carbamoyl-2-methylphenyl)-1H-pyrrol-1-yl)propanoic    acid;-   3-(2-(4-carbamoyl-2-methylphenyl)-1-(4-methoxyphenyl)-1H-pyrrol-3-yl)propanoic    acid;-   3-(2-(4-carbamoyl-2-methylphenyl)-3-(4-methoxyphenyl)-1H-pyrrol-1-yl)butanoic    acid;-   1-(2-(4-carbamoyl-2-methylphenyl)-3-(4-methoxyphenyl)-1H-pyrrol-1-yl)methyl)cyclopropanecarboxylic    acid;-   3-(2-(4-carbamoyl-2-methylphenyl)-3-(4-methoxyphenyl)-1H-pyrrol-1-yl)-2,2-difluoropropanoic    acid;-   3-(2-(4-carbamoyl-2-methylphenyl)-3-(4-methoxyphenyl)-1H-pyrrol-1-yl)-2-hydroxypropanoic    acid;-   3-(2-(4-carbamoyl-2-methylphenyl)-3-(4-methoxyphenyl)-1H-pyrrol-1-yl)hexanoic    acid;-   3-(2-(4-carbamoyl-2-methylphenyl)-3-(4-methoxyphenyl)-1H-pyrrol-1-yl)-4-methylpentanoic    acid;-   3-(1-(4-(1H-imidazol-1-yl)phenyl)-2-(4-carbamoyl-2-methylphenyl)-1H-pyrrol-3-yl)propanoic    acid;-   3-(2-(4-carbamoyl-2-methylphenyl)-1-(4-chloro-2-methoxyphenyl)-1H-pyrrol-3-yl)propanoic    acid;-   4-(2-(4-carbamoyl-2-methylphenyl)-3-(4-methoxyphenyl)-1H-pyrrol-1-yl)-2-hydroxybenzoic    acid;-   4-(2-(4-carbamoyl-2-methylphenyl)-3-(4-methoxyphenyl)-1H-pyrrol-1-yl)-2-fluorobenzoic    acid;-   4-(1-(4-hydroxy-3-nitrophenyl)-3-(4-methoxyphenyl)-1H-pyrrol-2-yl)-3-methylbenzamide;-   4-(1-(6-hydroxypyridin-3-yl)-3-(4-methoxyphenyl)-1H-pyrrol-2-yl)-3-methylbenzamide;-   4-(2-(4-carbamoyl-2-methylphenyl)-3-(4-methoxyphenyl)-1H-pyrrol-1-yl)benzoic    acid;-   4-(2-(4-carbamoyl-2-methylphenyl)-3-(4-methoxyphenyl)-1H-pyrrol-1-yl)-2-chlorobenzoic    acid;-   4-(3-(4-methoxyphenyl)-1-(2-(trifluoromethylsulfonamido)ethyl)-1H-pyrrol-2-yl)-3-methylbenzamide;-   4-(3-(4-methoxyphenyl)-1-(4-sulfamoylphenyl)-1H-pyrrol-2-yl)-3-methylbenzamide;-   4-(1-(3-fluoro-4-hydroxyphenyl)-3-(4-methoxyphenyl)-1H-pyrrol-2-yl)-3-methylbenzamide;-   5-(2-(4-carbamoyl-2-methylphenyl)-3-(4-methoxyphenyl)-1H-pyrrol-1-yl)picolinic    acid;-   4-(1-(3,5-difluoro-4-hydroxyphenyl)-3-(4-methoxyphenyl)-1H-pyrrol-2-yl)-3-methylbenzamide;-   4-(1-(5-hydroxypyridin-2-yl)-3-(4-methoxyphenyl)-1H-pyrrol-2-yl)-3-methylbenzamide;-   5-(2-(4-carbamoyl-2-methylphenyl)-3-(4-methoxyphenyl)-1H-pyrrol-1-yl)-2-hydroxybenzoic    acid;-   6-(2-(4-carbamoyl-2-methylphenyl)-3-(4-methoxyphenyl)-1H-pyrrol-1-yl)nicotinic    acid;-   4-(1-(3-fluoro-4-hydroxyphenyl)-3-(4-(2-methyl-1H-imidazol-1-yl)phenyl)-1H-pyrrol-2-yl)-3-methylbenzamide;    and-   4-(1-(3,5-difluoro-4-hydroxyphenyl)-3-(4-(2-methyl-1H-imidazol-1-yl)phenyl)-1H-pyrrol-2-yl)-3-methylbenzamide.

When a bond to a substituent is shown to cross a bond connecting twoatoms in a ring, then such substituent may be bonded to any atom in thering. When a substituent is listed without indicating the atom via whichsuch substituent is bonded to the rest of the compound of a givenformula, then such substituent may be bonded via any atom in suchsubstituent. Combinations of substituents and/or variables arepermissible, but only if such combinations result in stable compounds.

The compounds described herein may have asymmetric centers. Compounds ofthe present invention containing an asymmetrically substituted atom maybe isolated in optically active or racemic forms. It is well known inthe art how to prepare optically active forms, such as by resolution ofracemic forms or by synthesis from optically active starting materials.Many geometric isomers of olefins, C═N double bonds, and the like canalso be present in the compounds described herein, and all such stableisomers are contemplated in the present invention. Cis and transgeometric isomers of the compounds of the present invention aredescribed and may be isolated as a mixture of isomers or as separatedisomeric forms. All chiral, diastereomeric, racemic, and geometricisomeric forms of a structure are intended, unless the specificstereochemistry or isomeric form is specifically indicated. Alltautomers of shown or described compounds are also considered to be partof the present invention.

It is to be understood that isomers arising from such asymmetry (e.g.,all enantiomers and diastereomers) are included within the scope of theinvention, unless indicated otherwise. Such isomers can be obtained insubstantially pure form by classical separation techniques and bystereochemically controlled synthesis. Furthermore, the structures andother compounds and moieties discussed in this application also includeall tautomers thereof. Alkenes can include either the E- or Z-geometry,where appropriate.

2. Representative GSNOR Inhibitors

Examples 1-30 list representative novel pyrrole analogs of Formula Iuseful as GSNOR inhibitors of the invention. The synthetic methods thatcan be used to prepare each compound are detailed in Examples 1-30, withreference to synthetic schemes and intermediates described in Example31. Supporting mass spectrometry data and proton NMR data for eachcompound is also included in Examples 1-30. GSNOR inhibitor activity wasdetermined by the assay described in Example 32 and IC₅₀ values wereobtained. GSNOR inhibitor compounds in Examples 1-30 had an IC₅₀ ofabout <100 μM. GSNOR inhibitor compounds Examples 1, 3, 5, 6, 9, 13-17,19-20, 23, 25-27, 29-30 had an IC₅₀ of about <5 μM. GSNOR inhibitorcompounds Examples 1, 5-6, 13-15, 17, 19, 23, 25, 27, 29-30 had an IC₅₀of about less than 1.0 μM.

D. Pharmaceutical Compositions Comprising a GSNOR Inhibitor

The invention encompasses pharmaceutical compositions comprising atleast one GSNOR inhibitor described herein and at least onepharmaceutically acceptable carrier. Suitable carriers are described in“Remington: The Science and Practice, Twentieth Edition,” published byLippincott Williams & Wilkins, which is incorporated herein byreference. Pharmaceutical compositions according to the invention mayalso comprise one or more non-GSNOR inhibitor active agents.

The pharmaceutical compositions of the invention can comprise novelGSNOR inhibitors described herein, the pharmaceutical compositions cancomprise known compounds which previously were not know to have GSNORinhibitor activity, or a combination thereof.

The GSNOR inhibitors can be utilized in any pharmaceutically acceptabledosage form, including but not limited to injectable dosage forms,liquid dispersions, gels, aerosols, ointments, creams, lyophilizedformulations, dry powders, tablets, capsules, controlled releaseformulations, fast melt formulations, delayed release formulations,extended release formulations, pulsatile release formulations, mixedimmediate release and controlled release formulations, etc.Specifically, the GSNOR inhibitors described herein can be formulated:(a) for administration selected from the group consisting of oral,pulmonary, intravenous, intra-arterial, intrathecal, intra-articular,rectal, ophthalmic, colonic, parenteral, intracisternal, intravaginal,intraperitoneal, local, buccal, nasal, and topical administration; (b)into a dosage form selected from the group consisting of liquiddispersions, gels, aerosols, ointments, creams, tablets, sachets andcapsules; (c) into a dosage form selected from the group consisting oflyophilized formulations, dry powders, fast melt formulations,controlled release formulations, delayed release formulations, extendedrelease formulations, pulsatile release formulations, and mixedimmediate release and controlled release formulations; or (d) anycombination thereof.

For respiratory infections, an inhalation formulation can be used toachieve high local concentrations. Formulations suitable for inhalationinclude dry power or aerosolized or vaporized solutions, dispersions, orsuspensions capable of being dispensed by an inhaler or nebulizer intothe endobronchial or nasal cavity of infected patients to treat upperand lower respiratory bacterial infections.

Solutions or suspensions used for parenteral, intradermal, orsubcutaneous application can comprise one or more of the followingcomponents: (1) a sterile diluent such as water for injection, salinesolution, fixed oils, polyethylene glycols, glycerine, propylene glycolor other synthetic solvents; (2) antibacterial agents such as benzylalcohol or methyl parabens; (3) antioxidants such as ascorbic acid orsodium bisulfite; (4) chelating agents such asethylenediaminetetraacetic acid; (5) buffers such as acetates, citratesor phosphates; and (5) agents for the adjustment of tonicity such assodium chloride or dextrose. The pH can be adjusted with acids or bases,such as hydrochloric acid or sodium hydroxide. A parenteral preparationcan be enclosed in ampoules, disposable syringes or multiple dose vialsmade of glass or plastic.

Pharmaceutical compositions suitable for injectable use may comprisesterile aqueous solutions (where water soluble) or dispersions andsterile powders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. The pharmaceutical composition should bestable under the conditions of manufacture and storage and should bepreserved against the contaminating action of microorganisms such asbacteria and fungi.

The carrier can be a solvent or dispersion medium comprising, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), and suitablemixtures thereof. The proper fluidity can be maintained, for example, bythe use of a coating such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants. Prevention of the action of microorganisms can be achievedby various antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol or sorbitol, and inorganic saltssuch as sodium chloride in the composition. Prolonged absorption of theinjectable compositions can be brought about by including in thecomposition an agent which delays absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activereagent (e.g., GSNOR inhibitor) in the required amount in an appropriatesolvent with one or a combination of ingredients enumerated above, asrequired, followed by filtered sterilization. Generally, dispersions areprepared by incorporating at least one GSNOR inhibitor into a sterilevehicle that contains a basic dispersion medium and any other requiredingredients. In the case of sterile powders for the preparation ofsterile injectable solutions, exemplary methods of preparation includevacuum drying and freeze-drying, both of which yield a powder of theGSNOR inhibitor plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed, for example, in gelatin capsules orcompressed into tablets. For the purpose of oral therapeuticadministration, the GSNOR inhibitor can be incorporated with excipientsand used in the form of tablets, troches, or capsules. Oral compositionscan also be prepared using a fluid carrier for use as a mouthwash,wherein the compound in the fluid carrier is applied orally and swishedand expectorated or swallowed. Pharmaceutically compatible bindingagents, and/or adjuvant materials can be included as part of thecomposition.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser thatcontains a suitable propellant, e.g., a gas such as carbon dioxide, anebulized liquid, or a dry powder from a suitable device. Fortransmucosal or transdermal administration, penetrants appropriate tothe barrier to be permeated are used in the formulation. Such penetrantsare generally known in the art, and include, for example, fortransmucosal administration, detergents, bile salts, and fusidic acidderivatives. Transmucosal administration can be accomplished through theuse of nasal sprays or suppositories. For transdermal administration,the active reagents are formulated into ointments, salves, gels, orcreams as generally known in the art. The reagents can also be preparedin the form of suppositories (e.g., with conventional suppository basessuch as cocoa butter and other glycerides) or retention enemas forrectal delivery.

In one embodiment, the GSNOR inhibitors are prepared with carriers thatwill protect against rapid elimination from the body. For example, acontrolled release formulation can be used, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc.

Liposomal suspensions (including liposomes targeted to infected cellswith monoclonal antibodies to viral antigens) can also be used aspharmaceutically acceptable carriers. These can be prepared according tomethods known to those skilled in the art, for example, as described inU.S. Pat. No. 4,522,811.

Additionally, suspensions of the GSNOR inhibitors may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils, such as sesame oil, or synthetic fatty acidesters, such as ethyl oleate, triglycerides, or liposomes. Non-lipidpolycationic amino polymers may also be used for delivery. Optionally,the suspension may also include suitable stabilizers or agents toincrease the solubility of the compounds and allow for the preparationof highly concentrated solutions.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of GSNORinhibitor calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the GSNOR inhibitor and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active agent for thetreatment of individuals.

Pharmaceutical compositions according to the invention comprising atleast one GSNOR inhibitor can comprise one or more pharmaceuticalexcipients. Examples of such excipients include, but are not limited tobinding agents, filling agents, lubricating agents, suspending agents,sweeteners, flavoring agents, preservatives, buffers, wetting agents,disintegrants, effervescent agents, and other excipients. Suchexcipients are known in the art. Exemplary excipients include: (1)binding agents which include various celluloses and cross-linkedpolyvinylpyrrolidone, microcrystalline cellulose, such as Avicel® PH101and Avicel PH102, silicified microcrystalline cellulose (ProSolv SMCC™),gum tragacanth and gelatin; (2) filling agents such as various starches,lactose, lactose monohydrate, and lactose anhydrous; (3) disintegratingagents such as alginic acid, Primogel, corn starch, lightly crosslinkedpolyvinyl pyrrolidone, potato starch, maize starch, and modifiedstarches, croscarmellose sodium, cross-povidone, sodium starchglycolate, and mixtures thereof; (4) lubricants, including agents thatact on the flowability of a powder to be compressed, include magnesiumstearate, colloidal silicon dioxide, such as Aerosil® 200, talc, stearicacid, calcium stearate, and silica gel; (5) glidants such as colloidalsilicon dioxide; (6) preservatives, such as potassium sorbate,methylparaben, propylparaben, benzoic acid and its salts, other estersof parahydroxybenzoic acid such as butylparaben, alcohols such as ethylor benzyl alcohol, phenolic compounds such as phenol, or quaternarycompounds such as benzalkonium chloride; (7) diluents such aspharmaceutically acceptable inert fillers, such as microcrystallinecellulose, lactose, dibasic calcium phosphate, saccharides, and/ormixtures of any of the foregoing; examples of diluents includemicrocrystalline cellulose, such as Avicel® PH101 and Avicel® PH102;lactose such as lactose monohydrate, lactose anhydrous, and Pharmatose®DCL21; dibasic calcium phosphate such as Emcompress®; mannitol; starch;sorbitol; sucrose; and glucose; (8) sweetening agents, including anynatural or artificial sweetener, such as sucrose, saccharin sucrose,xylitol, sodium saccharin, cyclamate, aspartame, and acesulfame; (9)flavoring agents, such as peppermint, methyl salicylate, orangeflavoring, Magnasweet® (trademark of MAFCO), bubble gum flavor, fruitflavors, and the like; and (10) effervescent agents, includingeffervescent couples such as an organic acid and a carbonate orbicarbonate. Suitable organic acids include, for example, citric,tartaric, malic, fumaric, adipic, succinic, and alginic acids andanhydrides and acid salts. Suitable carbonates and bicarbonates include,for example, sodium carbonate, sodium bicarbonate, potassium carbonate,potassium bicarbonate, magnesium carbonate, sodium glycine carbonate,L-lysine carbonate, and arginine carbonate. Alternatively, only thesodium bicarbonate component of the effervescent couple may be present.

E. Kits Comprising the Compositions of the Invention

The present invention also encompasses kits comprising the compositionsof the invention. Such kits can comprise, for example, (1) at least oneGSNOR inhibitor; and (2) at least one pharmaceutically acceptablecarrier, such as a solvent or solution. Additional kit components canoptionally include, for example: (1) any of the pharmaceuticallyacceptable excipients identified herein, such as stabilizers, buffers,etc., (2) at least one container, vial or similar apparatus for holdingand/or mixing the kit components; and (3) delivery apparatus, such as aninhaler, nebulizer, syringe, etc.

F. Methods of Preparing GSNOR Inhibitors

The GSNOR inhibitors of the invention can readily be synthesized usingknown synthetic methodologies or via a modification of known syntheticmethodologies. As would be readily recognized by a skilled artisan, themethodologies described below allow the synthesis of pyrroles having avariety of substituents. Exemplary synthetic methods are described inthe examples below.

If needed, further purification and separation of enantiomers anddiastereomers can be achieved by routine procedures known in the art.Thus, for example, the separation of enantiomers of a compound can beachieved by the use of chiral HPLC and related chromatographictechniques. Diastereomers can be similarly separated. In some instances,however, diastereomers can simply be separated physically, such as, forexample, by controlled precipitation or crystallization.

The process of the invention, when carried out as prescribed herein, canbe conveniently performed at temperatures that are routinely accessiblein the art. In one embodiment, the process is performed at a temperaturein the range of about 25° C. to about 110° C. In another embodiment, thetemperature is in the range of about 40° C. to about 100° C. In yetanother embodiment, the temperature is in the range of about 50° C. toabout 95° C.

Synthetic steps that require a base are carried out using any convenientorganic or inorganic base. Typically, the base is not nucleophilic.Thus, in one embodiment, the base is selected from carbonates,phosphates, hydroxides, alkoxides, salts of disilazanes, and tertiaryamines.

The process of the invention, when performed as described herein, can besubstantially complete after several minutes to after several hoursdepending upon the nature and quantity of reactants and reactiontemperature. The determination of when the reaction is substantiallycomplete can be conveniently evaluated by ordinary techniques known inthe art such as, for example, HPLC, LCMS, TLC, and ¹H NMR.

G. Method of Treatment

The invention encompasses methods of preventing or treating (e.g.,alleviating one or more symptoms of) medical conditions through use ofone or more of the disclosed compounds. The methods compriseadministering a therapeutically effective amount of a GSNOR inhibitor toa patient in need. The compositions of the invention can also be usedfor prophylactic therapy.

The GSNOR inhibitor used in the methods of treatment according to theinvention can be: (1) a novel GSNOR inhibitor described herein, or apharmaceutically acceptable salt thereof, a prodrug thereof, or ametabolite thereof; (2) a compound which was known prior to the presentinvention, but wherein it was not known that the compound is a GSNORinhibitor, or a pharmaceutically acceptable salt thereof, a prodrugthereof, or a metabolite thereof; or (3) a compound which was knownprior to the present invention, and wherein it was known that thecompound is a GSNOR inhibitor, but wherein it was not known that thecompound is useful for the methods of treatment described herein, or apharmaceutically acceptable salt thereof, a prodrug thereof, or ametabolite thereof.

The patient can be any animal, domestic, livestock or wild, including,but not limited to cats, dogs, horses, pigs and cattle, and preferablyhuman patients. As used herein, the terms patient and subject may beused interchangeably.

In subjects with deleteriously high levels of GSNOR or GSNOR activity,modulation may be achieved, for example, by administering one or more ofthe disclosed compounds that disrupts or down-regulates GSNOR function,or decreases GSNOR levels. These compounds may be administered withother GSNOR inhibitor agents, such as anti-GSNOR antibodies or antibodyfragments, GSNOR antisense, iRNA, or small molecules, or otherinhibitors, alone or in combination with other agents as described indetail herein.

The present invention provides a method of treating a subject afflictedwith a disorder ameliorated by NO donor therapy. Such a method comprisesadministering to a subject a therapeutically effective amount of a GSNORinhibitor.

As used herein, “treating” describes the management and care of apatient for the purpose of combating a disease, condition, or disorderand includes the administration of a compound of the present inventionto prevent the onset of the symptoms or complications, alleviating thesymptoms or complications, or eliminating the disease, condition ordisorder. More specifically, “treating” includes reversing, attenuating,alleviating, minimizing, suppressing or halting at least one deleterioussymptom or effect of a disease (disorder) state, disease progression,disease causative agent (e.g., bacteria or viruses), or other abnormalcondition. Treatment is continued as long as symptoms and/or pathologyameliorate.

The disorders can include pulmonary disorders associated with hypoxemiaand/or smooth muscle constriction in the lungs and/or lung infectionand/or lung injury (e.g., pulmonary hypertension, ARDS, asthma,pneumonia, pulmonary fibrosis/interstitial lung diseases, cysticfibrosis COPD) cardiovascular disease and heart disease, includingconditions such as hypertension, ischemic coronary syndromes,atherosclerosis, heart failure, glaucoma, diseases characterized byangiogenesis (e.g., coronary artery disease), disorders where there isrisk of thrombosis occurring, disorders where there is risk ofrestenosis occurring, chronic inflammatory diseases (e.g., AID dementiaand psoriasis), diseases where there is risk of apoptosis occurring(e.g., heart failure, atherosclerosis, degenerative neurologicdisorders, arthritis and liver injury (ischemic or alcoholic)),impotence, obesity caused by eating in response to craving for food,stroke, reperfusion injury (e.g., traumatic muscle injury in heart orlung or crush injury), and disorders where preconditioning of heart orbrain for NO protection against subsequent ischemic events isbeneficial.

In one embodiment, the compounds of the present invention or apharmaceutically acceptable salt thereof, or a prodrug or metabolitethereof, can be administered in combination with an NO donor. An NOdonor donates nitric oxide or a related redox species and more generallyprovides nitric oxide bioactivity, that is activity which is identifiedwith nitric oxide, e.g., vasorelaxation or stimulation or inhibition ofa receptor protein, e.g., ras protein, adrenergic receptor, NFκB. NOdonors including S-nitroso, O-nitroso, C-nitroso and N-nitroso compoundsand nitro derivatives thereof and metal NO complexes, but not excludingother NO bioactivity generating compounds, useful herein are describedin “Methods in Nitric Oxide Research,” Feelisch et al. eds., pages71-115 (J. S., John Wiley & Sons, New York, 1996), which is incorporatedherein by reference. NO donors which are C-nitroso compounds wherenitroso is attached to a tertiary carbon which are useful herein includethose described in U.S. Pat. No. 6,359,182 and in WO 02/34705. Examplesof S-nitroso compounds, including S-nitrosothiols useful herein,include, for example, S-nitrosoglutathione,S-nitroso-N-acetylpenicillamine, S-nitroso-cysteine and ethyl esterthereof, S-nitroso cysteinyl glycine,S-nitroso-gamma-methyl-L-homocysteine, S-nitroso-L-homocysteine,S-nitroso-gamma-thio-L-leucine, S-nitroso-delta-thio-L-leucine, andS-nitrosoalbumin. Examples of other NO donors useful herein are sodiumnitroprusside (nipride), ethyl nitrite, isosorbide, nitroglycerin, SIN 1which is molsidomine, furoxamines, N-hydroxy (N-nitrosamine) andperfluorocarbons that have been saturated with NO or a hydrophobic NOdonor.

The combination of a GSNOR inhibitor with R(+) enantiomer of amlodipine,a known NO releaser (Zhang X. P at al. 2002 J. CardiovascularPharmacology 39, 208-214) is also an embodiment of the presentinvention.

The present invention also provides a method of treating a subjectafflicted with pathologically proliferating cells where the methodcomprises administering to said subject a therapeutically effectiveamount of an inhibitor of GSNOR. The inhibitors of GSNOR are thecompounds as defined above, or a pharmaceutically acceptable saltthereof, or a prodrug or metabolite thereof, in combination with apharmaceutically acceptable carrier. Treatment is continued as long assymptoms and/or pathology ameliorate.

In another embodiment, the pathologically proliferating cells can bepathologically proliferating microbes. The microbes involved can bethose where GSNOR is expressed to protect the microbe from nitrosativestress or where a host cell infected with the microbe expresses theenzyme, thereby protecting the microbe from nitrosative stress. The term“pathologically proliferating microbes” is used herein to meanpathologic microorganisms including but not limited to pathologicbacteria, pathologic viruses, pathologic Chlamydia, pathologic protozoa,pathologic Rickettsia, pathologic fungi, and pathologic mycoplasmata.More detail on the applicable microbes is set forth at columns 11 and 12of U.S. Pat. No. 6,057,367. The term “host cells infected withpathologic microbes” includes not only mammalian cells infected withpathologic viruses but also mammalian cells containing intracellularbacteria or protozoa, e.g., macrophages containing Mycobacteriumtuberculosis, Mycobacterium leper (leprosy), or Salmonella typhi(typhoid fever).

In another embodiment, the pathologically proliferating cells can bepathologic helminths. The term “pathologic helminths” is used herein torefer to pathologic nematodes, pathologic trematodes and pathologiccestodes. More detail on the applicable helminths is set forth at column12 of U.S. Pat. No. 6,057,367.

In another embodiment, the pathologically proliferating cells can bepathologically proliferating mammalian cells. The term “pathologicallyproliferating mammalian cells” as used herein means cells of the mammalthat grow in size or number in said mammal so as to cause a deleteriouseffect in the mammal or its organs. The term includes, for example, thepathologically proliferating or enlarging cells causing restenosis, thepathologically proliferating or enlarging cells causing benign prostatichypertrophy, the pathologically proliferating cells causing myocardialhypertrophy and proliferating cells at inflammatory sites such assynovial cells in arthritis or cells associated with a cellproliferation disorder.

As used herein, the term “cell proliferative disorder” refers toconditions in which the unregulated and/or abnormal growth of cells canlead to the development of an unwanted condition or disease, which canbe cancerous or non-cancerous, for example a psoriatic condition. Asused herein, the term “psoriatic condition” refers to disordersinvolving keratinocyte hyperproliferation, inflammatory cellinfiltration, and cytokine alteration. The cell proliferative disordercan be a precancerous condition or cancer. The cancer can be primarycancer or metastatic cancer, or both.

As used herein, the term “cancer” includes solid tumors, such as lung,breast, colon, ovarian, pancreas, prostate, adenocarcinoma, squamouscarcinoma, sarcoma, malignant glioma, leiomyosarcoma, hepatoma, head andneck cancer, malignant melanoma, non-melanoma skin cancers, as well ashematologic tumors and/or malignancies, such as leukemia, childhoodleukemia and lymphomas, multiple myeloma, Hodgkin's disease, lymphomasof lymphocytic and cutaneous origin, acute and chronic leukemia such asacute lymphoblastic, acute myelocytic or chronic myelocytic leukemia,plasma cell neoplasm, lymphoid neoplasm and cancers associated withAIDS.

In addition to psoriatic conditions, the types of proliferative diseaseswhich may be treated using the compositions of the present invention areepidermic and dermoid cysts, lipomas, adenomas, capillary and cutaneoushemangiomas, lymphangiomas, nevi lesions, teratomas, nephromas,myofibromatosis, osteoplastic tumors, and other dysplastic masses andthe like. In one embodiment, proliferative diseases include dysplasiasand disorders of the like.

In one embodiment, the treating cancer comprises a reduction in tumorsize, decrease in tumor number, a delay of tumor growth, decrease inmetastaic lesions in other tissues or organs distant from the primarytumor site, an improvement in the survival of patients, or animprovement in the quality of patient life, or at least two of theabove.

In another embodiment, the treating a cell proliferative disordercomprises a reduction in the rate of cellular proliferation, reductionin the proportion of proliferating cells, a decrease in size of an areaor zone of cellular proliferation, or a decrease in the number orproportion of cells having an abnormal appearance or morphology, or atleast two of the above.

In yet another embodiment, the compounds of the present invention or apharmaceutically acceptable salt thereof, a prodrug thereof, ormetabolite thereof, can be administered in combination with a secondchemotherapeutic agent. In a further embodiment, the secondchemotherapeutic agent is selected from the group consisting oftamoxifen, raloxifene, anastrozole, exemestane, letrozole, cisplatin,carboplatin, paclitaxel, cyclophosphamide, lovastatin, minosine,gemcitabine, araC, 5-fluorouracil, methotrexate, docetaxel, goserelin,vincristin, vinblastin, nocodazole, teniposide, etoposide, epothilone,navelbine, camptothecin, daunonibicin, dactinomycin, mitoxantrone,amsacrine, doxorubicin, epirubicin, idarubicin imatanib, gefitinib,erlotinib, sorafenib, sunitinib malate, trastuzumab, rituximab,cetuximab, and bevacizumab.

In one embodiment, the compounds of the present invention or apharmaceutically acceptable salt thereof, a prodrug thereof, ormetabolite thereof, can be administered in combination with an agentthat imposes nitrosative or oxidative stress. Agents for selectivelyimposing nitrosative stress to inhibit proliferation of pathologicallyproliferating cells in combination therapy with GSNOR inhibitors hereinand dosages and routes of administration therefore include thosedisclosed in U.S. Pat. No. 6,057,367, which is incorporated herein.Supplemental agents for imposing oxidative stress (i.e., agents thatincrease GSSG (oxidized glutathione) over GSH (glutathione) ratio orNAD(P) over NAD(P)H ratio or increase thiobarbituric acid derivatives)in combination therapy with GS-FDH inhibitors herein include, forexample, L-buthionine-5-sulfoximine (BSO), glutathione reductaseinhibitors (e.g., BCNU), inhibitors or uncouplers of mitochondrialrespiration and drugs that increase reactive oxygen species (ROS), e.g.,adriamycin, in standard dosages with standard routes of administration.

GSNOR inhibitors may also be co-administered with a phosphodiesteraseinhibitor (e.g., rolipram, cilomilast, roflumilast, Viagra® (sildenifilcitrate), Cialis® (tadalafil), Levitra® (vardenifil), etc.), aβ-agonist, a steroid, or a leukotriene antagonist (LTD-4). Those skilledin the art can readily determine the appropriate therapeuticallyeffective amount depending on the disorder to be ameliorated.

GSNOR inhibitors may be used as a means to improve β-adrenergicsignaling. In particular, inhibitors of GSNOR alone or in combinationwith β-agonists could be used to treat or protect against heart failure,or other vascular disorders such as hypertension and asthma. GSNORinhibitors can also be used to modulate G protein coupled receptors(GPCRs) by potentiating Gs G-protein, leading to smooth musclerelaxation (e.g., airway and blood vessels), and by attenuating GqG-protein, and thereby preventing smooth muscle contraction (e.g., inairway and blood vessels).

The therapeutically effective amount for the treatment of a subjectafflicted with a disorder ameliorated by NO donor therapy is the GSNORinhibiting amount in vivo that causes amelioration of the disorder beingtreated or protects against a risk associated with the disorder. Forexample, for asthma, a therapeutically effective amount is abronchodilating effective amount; for cystic fibrosis, a therapeuticallyeffective amount is an airway obstruction ameliorating effective amount;for ARDS, a therapeutically effective amount is a hypoxemia amelioratingeffective amount; for heart disease, a therapeutically effective amountis an angina relieving or angiogenesis inducing effective amount; forhypertension, a therapeutically effective amount is a blood pressurereducing effective amount; for ischemic coronary disorders, atherapeutic amount is a blood flow increasing effective amount; foratherosclerosis, a therapeutically effective amount is an endothelialdysfunction reversing effective amount; for glaucoma, a therapeuticamount is an intraocular pressure reducing effective amount; fordiseases characterized by angiogenesis, a therapeutically effectiveamount is an angiogenesis inhibiting effective amount; for disorderswhere there is risk of thrombosis occurring, a therapeutically effectiveamount is a thrombosis preventing effective amount; for disorders wherethere is risk of restenosis occurring, a therapeutically effectiveamount is a restenosis inhibiting effective amount; for chronicinflammatory diseases, a therapeutically effective amount is aninflammation reducing effective amount; for disorders where there isrisk of apoptosis occurring, a therapeutically effective amount is anapoptosis preventing effective amount; for impotence, a therapeuticallyeffective is an erection attaining or sustaining effective amount; forobesity, a therapeutically effective amount is a satiety causingeffective amount; for stroke, a therapeutically effective amount is ablood flow increasing or a TIA protecting effective amount; forreperfusion injury, a therapeutically effective amount is a functionincreasing effective amount; and for preconditioning of heart and brain,a therapeutically effective amount is a cell protective effectiveamount, e.g., as measured by triponin or CPK.

The therapeutically effective amount for the treatment of a subjectafflicted with pathologically proliferating cells means a GSNORinhibiting amount in vivo which is an antiproliferative effectiveamount. Such antiproliferative effective amount as used herein means anamount causing reduction in rate of proliferation of at least about 20%,at least about 10%, at least about 5%, or at least about 1%.

In general, the dosage, i.e., the therapeutically effective amount,ranges from 1 μg to 10 g/kg and often ranges from 10 μg to 1 g/kg or 10μg to 100 mg/kg body weight of the subject being treated, per day.

H. Other Uses

The compounds of the present invention or a pharmaceutically acceptablesalt thereof, or a prodrug or metabolite thereof, can be applied tovarious apparatus in circumstances when the presence of such compoundswould be beneficial. Such apparatus can be any device or container, forexample, implantable devices in which a GSNOR inhibitor can be used tocoat a surgical mesh or cardiovascular stent prior to implantation in apatient. The GSNOR inhibitors of the present invention can also beapplied to various apparatus for in vitro assay purposes or forculturing cells.

The compounds of the present invention or a pharmaceutically acceptablesalt thereof, or a prodrug or metabolite thereof, can also be used as anagent for the development, isolation or purification of binding partnersto GSNOR inhibitor compounds, such as antibodies, natural ligands, andthe like. Those skilled in the art can readily determine related usesfor the compounds of the present invention.

EXAMPLES

The following examples are given to illustrate the present invention. Itshould be understood, however, that the invention is not to be limitedto the specific conditions or details described in these examples.Throughout the specification, any and all references to a publiclyavailable document, including a U.S. patent, are specificallyincorporated by reference.

Examples 1-30 list representative novel pyrrole analogs of Formula Iuseful as GSNOR inhibitors of the invention. The synthetic methods thatcan be used to prepare each compound are detailed in Examples 1-30, withreference to synthetic schemes described in Example 31.

Example 13-(2-(4-carbamoyl-2-methylphenyl)-3-(4-methoxyphenyl)-1H-pyrrol-1-yl)propanoicacid

Synthesis:

Scheme 1, R=propanoic acid-3-yl with minor deviations. In step 1, thereaction was stirred at room temperature overnight and then purified bypreparative TLC. In step 2, the mixture was stirred at room temperatureovernight. Water was added and the mixture was washed with diethylether, the aqueous layer was adjusted to PH=4-5 with 1 N hydrochloricacid. The resulting mixture was extracted with ethyl acetate (30 mL×4).The combined organic layer was washed with brine, dried over sodiumsulfate, and evaporated. The residue was purified on silica gel column(eluting with 5% methanol in dichloromethane), followed by preparativeHPLC to give the title compound.

Characterization Data:

¹H NMR (DMSO-d₆, 400 MHz TMS): δ 8.01 (brs, 1H), 7.79 (s, 1H), 7.77 (d,J=8.0 Hz, 1H), 7.39 (brs, 1H), 7.35 (d, J=8.0 Hz, 1H), 6.93 (m, 3H),6.70 (d, J=8.8 Hz, 2H), 6.35 (d, J=2.8 Hz, 1H), 3.88 (m, 1H), 3.74 (m,1H), 3.63 (s, 3H), 2.42 (t, J=7.2 Hz, 2H), 1.91 (s, 3H); MS (ESI): m/z379.1 [M+1]⁺.

Example 23-(2-(4-carbamoyl-2-methylphenyl)-3-(4-methoxyphenyl)-1H-pyrrol-1-yl)-3-phenylpropanoicacid

Synthesis:

Scheme 1, R=3-phenylpropanoic acid-3-yl

Characterization Data:

¹H NMR (CD₃OD 400 MHz TMS): δ 7.76 (d, J=8.0 Hz, 0.5; H), 7.68 (s, 0.4;H), 7.54 (d, J=8.0 Hz, 0.6H), 7.43 (s, 0.5H), 7.35 (d, J=8.0 Hz, 0.5H),7.19-7.13 (m, 2H), 7.08-7.04 (m, 2H), 6.88-6.85 (m, 3H), 6.74-6.71 (m,1H), 6.61 (d, J=8 Hz, 0.4H), 6.55 (d, J=8.8 Hz, 2H), 6.41 (d, J=2.8 Hz,0.4H), 6.36 (d, J=2.8 Hz, 0.5H), 5.42-5.38 (m, 0.6H), 5.14-5.10 (m,0.4H), 3.58 (s, 3H), 3.28-3.24 (m, 0.5H), 3.11-3.03 (m, 0.5H), 3.00-2.93(m, 1H), 1.99 (s, 1.3H), 1.21 (s, 1.7H); MS (ESI): m/z 477.1 [M+23]⁺.

Example 33-(2-(4-carbamoyl-2-methylphenyl)-3-(4-methoxyphenyl)-1H-pyrrol-1-yl)-2-methylpropanoicacid

Synthesis:

Scheme 1, R=Scheme 1, R=2-methylpropanoic acid-3-yl

Characterization Data:

¹H NMR (CD₃OD 400 MHz TMS): δ 7.80 (d, J=7.2 Hz, 2H), 7.40 (d, J=7.6 Hz,1H), 6.98 (d, J=8.8 Hz, 2H), 6.88 (dd, J₁=1.6 Hz, J₂=5.6 Hz, 1H), 6.69(d, J=8.4 Hz, 2H), 6.37 (dd, J₁=2.8 Hz, J₂=7.2 Hz, 0.6H), 4.19-4.13 (m,0.6H), 3.99-3.93 (m, 0.4H), 3.86-3.81 (m, 0.5H), 3.72 (s, 3H), 3.62-3.57(m, 0.6H), 2.57-2.49 (m, 1H), 2.04 (s, 3H), 0.95 (dd, J=2.8 Hz, J=7.2Hz, 3H); MS (ESI): m/z 393.0 [M+1]⁺.

Example 44-(2-(4-carbamoyl-2-methylphenyl)-3-(4-methoxyphenyl)-1H-pyrrol-1-yl)butanoicacid

Synthesis:

Scheme 1, R=n-butanoic acid-4-yl

Characterization Data:

¹H NMR (G000003136 00125-061-1 CD₃OD 400 MHz TMS): δ 7.79 (s, 1H), 7.78(d, J=7.6 Hz, 1H), 7.37 (d, J=8.0 Hz, 1H), 6.99 (d, J=8.8 Hz, 2H), 6.88(d, J=2.8 Hz, 1H), 6.69 (d, J=8.8 Hz, 2H), 6.40 (d, J=2.8 Hz, 1H),3.90-3.84 (m, 1H), 3.66 (s, 3H), 3.39-3.63 (m, 1H), 2.13 (m, 2H), 2.03(m, 3H), 1.78-1.70 (m, 2H); MS (ESI): m/z 394.1 [M+1]⁺.

Example 53-(3-(4-(1H-imidazol-1-yl)phenyl)-2-(4-carbamoyl-2-methylphenyl)-1H-pyrrol-1-yl)propanoicacid

Synthesis:

Scheme 2, R1=propanoic acid-3-yl, R2=1H-imidazol-1-yl

Characterization Data:

¹H NMR (DMSO-d₆, 400 MHz): δ 9.33 (s, 1H), 8.11-8.06 (m, 2H), 7.87 (s,1H), 7.83 (d, J=8.4 Hz, 1H), 7.73 (s, 1H), 7.56-7.54 (d, J=8.4 Hz, 2H),7.44 (m, 3H), 7.21 (d, J=8.4 Hz, 2H), 7.05 (d, J=2.8 Hz, 1H), 6.57 (d,J=3.2 Hz, 1H), 3.93-3.88 (m, 2H), 3.79-3.74 (m, 2H), 2.09 (s, 3H); MS(ESI): m/z 415.3 [M+1]⁺.

Example 63-(2-(4-carbamoyl-2-methylphenyl)-1-(4-methoxyphenyl)-1H-pyrrol-3-yl)propanoicacid

Synthesis:

Scheme 4, R1=4-methoxyphenyl

Characterization Data:

¹H NMR (CDCl₃ 400 MHz TMS): δ 7.48 (d, J=8.2 Hz, 1H), 7.47 (s, 1H), 7.23(d, J=7.2 Hz, 2H), 6.80 (m, 3H), 6.63 (d, J=8.8 Hz, 2H), 3.66 (s, 3H),2.57 (m, 2H), 2.45 (m, 2H), 1.85 (s, 3H); MS (ESI): m/z 379.2 [M+1]⁺.

Example 73-(2-(4-carbamoyl-2-methylphenyl)-3-(4-methoxyphenyl)-1H-pyrrol-1-yl)butanoicacid

Synthesis:

Scheme 1, R=butanoic acid-3-yl

Characterization Data:

¹H NMR (CD₃OD 400 MHz TMS): δ 7.79-7.7 (m, 2H), 7.48 (d, J=8.4 Hz, 1H),6.99-6.96 (m, 3H), 6.67 (d, J=8.8 Hz, 2H), 6.42 (d, J=3.2 Hz, 1H),4.29-4.24 (m, 1H), 3.70 (s, 3H), 2.87-2.81 (m, 1H), 2.74-2.67 (m, 1H),2.00 (s, 3H), 1.30 (d, J=6.8 Hz, 3H); MS (ESI): m/z 393.1 [M+1]⁺.

Example 81-(2-(4-carbamoyl-2-methylphenyl)-3-(4-methoxyphenyl)-1H-pyrrol-1-yl)methyl)cyclopropanecarboxylicacid

Synthesis:

Scheme 1, R=1-methyl cyclopropanecarboxylic acid

Characterization Data:

¹H NMR (CD₃OD 300 MHz TMS): δ 7.76 (m, 2H), 7.36 (d, J=7.8 Hz, 1H), 7.05(d, J=3.0 Hz, 1H), 6.97 (d, J=9.0 Hz, 2H), 6.68 (d, J=9.0 Hz, 2H), 6.36(d, J=3.0 Hz, 1H), 3.95 (s, 2H), 3.71 (s, 3H), 2.01 (s, 3H), 1.19 (m,2H), 0.76-0.72 (m, 1H), 0.59-0.55 (m, 1H); MS (ESI): m/z 427.1 [M+23]⁺.

Example 93-(2-(4-carbamoyl-2-methylphenyl)-3-(4-methoxyphenyl)-1H-pyrrol-1-yl)-2,2-difluoropropanoicacid

Synthesis:

Scheme 1, R=2,2-difluoropropanoic acid-3-yl

Characterization Data:

¹H NMR (CD₃OD 400 MHz TMS): δ 7.80-7.78 (m, 2H), 7.38 (d, J=7.6 Hz, 1H),7.00-6.95 (m, 3H), 6.71-6.69 (m, 2H), 6.45 (d, J=2.8 Hz, 1H), 4.50-4.39(m, 1H), 4.31-4.20 (m, 1H), 3.72 (s, 3H), 1.97 (s, 3H); MS (ESI): m/z414.1 [M+1]⁺.

Example 103-(2-(4-carbamoyl-2-methylphenyl)-3-(4-methoxyphenyl)-1H-pyrrol-1-yl)-2-hydroxypropanoicacid

Synthesis:

Scheme 1, R=2-hydroxypropanoic acid-3-yl

Characterization Data:

¹H NMR (CD₃OD 400 MHz TMS): δ 7.78-7.76 (m, 2H), 7.44-7.39 (m, 1H),6.99-6.96 (m, 3H), 6.67 (d, J=8.4 Hz, 2H), 6.37 (d, J=3.2 Hz, 1H),4.20-4.12 (m, 1H), 4.07-4.04 (m, 0.5H), 4.00-3.89 (m, 1H), 3.81-3.75 (m,0.5H), 3.70 (s, 3H), 2.01 (m, 3H); MS (ESI): m/z 395.1 [M+1]⁺.

Example 113-(2-(4-carbamoyl-2-methylphenyl)-3-(4-methoxyphenyl)-1H-pyrrol-1-yl)hexanoicacid

Synthesis:

Scheme 1, R=hexanoic acid-3-yl

Characterization Data:

¹H NMR (CD₃OD 300 MHz TMS): δ 7.82 (dd, J=1.5 Hz, J=8.1 Hz, 1H), 7.74(s, 1H), 7.56 (d, J=7.8 Hz, 1H), 6.99-6.96 (m, 3H), 6.68 (d, J=8.7 Hz,2H), 6.43 (d, J=3.0 Hz, 1H), 4.27-4.17 (m, 1H), 3.71 (s, 3H), 2.94-2.75(m, 2H), 1.92 (s, 3H), 1.76-1.56 (m, 2H), 1.14-0.92 (m, 2H), 0.70 (t,J=7.2 Hz, 3H); MS (ESI): m/z=421.2 [M+1]⁺.

Example 123-(2-(4-carbamoyl-2-methylphenyl)-3-(4-methoxyphenyl)-1H-pyrrol-1-yl)-4-methylpentanoicacid

Synthesis:

Scheme 1, R=4-methylpentanoic acid-3-yl, with a deviation in step 1. 2equivalents of HOAc was used in place of PPTS and the reaction wasstirred at 120° C. under microwave irradiation for 1 hour. Mixture wasconcentrated in vacuo to afford the crude compound, which was useddirectly in the next step.

Characterization Data:

¹H NMR (CD₃OD 400 MHz TMS): δ 7.81 (d, J=7.6 Hz, 1H), 7.70-7.67 (m, 2H),6.96-6.94 (m, 3H), 6.68 (d, J=8.8 Hz, 2H), 6.39 (d, J=2.8 Hz, 1H),4.18-4.12 (m, 1H), 3.71 (s, 3H), 2.93-2.89 (m, 2H), 1.89-1.85 (m, 4H),0.79 (d, J=6.8 Hz, 3H), 0.60 (d, J=6.8 Hz, 3H); MS (ESI): m/z 443.1[M+23]⁺.

Example 133-(1-(4-(1H-imidazol-1-yl)phenyl)-2-(4-carbamoyl-2-methylphenyl)-1H-pyrrol-3-yl)propanoicacid

Synthesis:

Scheme 5, R1=(1H-imidazol-1-yl)phenyl.

Characterization Data:

¹H NMR (CD₃OD, 400 MHz TMS): δ 8.14 (brs, 1H), 7.74 (d, J=8.0 Hz, 1H),7.68 (s, 1H), 7.55 (brs, 1H), 7.49 (d, J=8.4 Hz, 2H), 7.42 (d, J=8.0 Hz,1H), 7.24 (d, J=8.4 Hz, 2H), 7.15 (s, 1H), 7.08 (d, J=2.0 Hz, 1H), 6.37(d, J=2.8 Hz, 1H), 2.64 (m, 2H), 2.49 (m, 2H), 1.97 (s, 3H); MS (ESI):m/z 415.2 [M+1]⁺.

Example 143-(2-(4-carbamoyl-2-methylphenyl)-1-(4-chloro-2-methoxyphenyl)-1H-pyrrol-3-yl)propanoicacid

Synthesis:

Scheme 3, R1=4-chloro-2-methoxyphenyl

Characterization Data:

¹H NMR (DMSO-d₆ 400 MHz TMS): δ 7.87 (s, 1H), 7.64 (s, 1H), 7.55 (d,J=7.6 Hz, 1H), 7.29 (s, 1H), 7.00-7.15 (m, 3H), 6.80-6.95 (m, 2H), 6.21(s, 1H), 3.60 (s, 3H), 2.37-2.42 (m, 4H), 2.00 (s, 3H); MS (ESI): m/z413.0 [M+1]⁺.

Example 154-(2-(4-carbamoyl-2-methylphenyl)-3-(4-methoxyphenyl)-1H-pyrrol-1-yl)-2-hydroxybenzoicacid

Synthesis:

Scheme 1, R=4-carboxy-3-hydroxyphenyl

Characterization Data:

¹H NMR (CD₃OD 400 MHz TMS): δ 7.62-7.58 (m, 2H), 7.52 (d, J=7.6 Hz, 1H),7.11-7.06 (m, 2H), 6.92 (d, J=8.8 Hz, 2H), 6.63 (d, J=8.8 Hz, 2H),6.50-6.47 (m, 3H), 3.62 (s, 3H), 1.85 (s, 3H); MS (ESI): m/z 443.0[M+1]⁺.

Example 164-(2-(4-carbamoyl-2-methylphenyl)-3-(4-methoxyphenyl)-1H-pyrrol-1-yl)-2-fluorobenzoicacid

Synthesis:

Scheme 1, R=4-carboxy-3-fluoro phenyl

Characterization Data:

¹H NMR (CD₃OD 400 MHz TMS): δ 7.84 (t, J=8 Hz, 1H), 7.73 (s, 1H), 7.67(d, J=7.6 Hz, 1H), 7.26-7.24 (m, 2H), 7.06 (d, J=8.8 Hz, 2H), 6.99-6.91(m, 2H), 6.77 (d, J=8.4 Hz, 2H), 6.65 (d, J=3.2 Hz, 1H), 3.76 (s, 3H),1.98 (s, 3H); MS (ESI): m/z 445.0 [M+1]⁺.

Example 174-(1-(4-hydroxy-3-nitrophenyl)-3-(4-methoxyphenyl)-1H-pyrrol-2-yl)-3-methylbenzamide

Synthesis:

Scheme 1, R=4-hydroxy-3-nitrophenyl

Characterization Data:

¹H NMR (CD₃OD 400 MHz TMS): δ 7.78 (t, J=2.4 Hz, 1H), 7.69 (s, 1H), 7.64(d, J=8.0 Hz, 1H), 7.4 (dd, J=2.8 Hz, J=8.8 Hz, 1H), 7.24 (d, J=7.6 Hz,1H), 7.13 (d, J=2.8 Hz, 1H), 7.07-7.03 (m, 3H), 6.74 (d, J=8.4 Hz, 2H),6.60 (d, J=3.2 Hz, 1H), 3.74 (s, 3H), 1.98 (s, 3H); MS (ESI): m/z 444.0[M+1]⁺.

Example 184-(1-(6-hydroxypyridin-3-yl)-3-(4-methoxyphenyl)-1H-pyrrol-2-yl)-3-methylbenzamide

Synthesis:

Scheme 1, R=6-hydroxypyridin-3-yl

Characterization Data:

¹H NMR (CD₃OD 400 MHz TMS): δ 7.72 (s, 1H), 7.68 (d, J=7.6 Hz, 1H), 7.43(dd, J=2.8 Hz, J=8.8 Hz, 1H), 7.35 (d, J=2.8 Hz, 1H), 7.28 (d, J=8.0 Hz,1H), 7.07-7.05 (m, 3H), 6.74 (d, J=8.8 Hz, 2H), 6.59 (d, J=2.8 Hz, 1H),6.46 (d, J=8.4 Hz, 1H), 3.74 (s, 3H), 2.00 (s, 3H); MS (ESI): m/z 400.0[M+1]⁺.

Example 194-(2-(4-carbamoyl-2-methylphenyl)-3-(4-methoxyphenyl)-1H-pyrrol-1-yl)benzoicacid

Synthesis:

Scheme 1, R=4-carboxyphenyl

Characterization Data:

¹H NMR (DMSO-d₆ 400 MHz TMS): δ 7.95 (s, 1H), 7.83 (d, J=8.8 Hz, 2H),7.67 (s, 1H), 7.63 (m, 1H), 7.37 (s, 1H), 7.29 (d, J=2.8 Hz, 1H), 7.22(d, J=7.6 Hz, 1H), 7.16 (d, J=8.8 Hz, 2H), 7.02 (d, J=8.8 Hz, 2H), 6.77(d, J=8.8 Hz, 2H), 6.63 (d, J=2.8 Hz, 1H), 3.68 (s, 3H), 1.85 (s, 3H);MS (ESI): m/z 427.2 [M+1]⁺.

Example 204-(2-(4-carbamoyl-2-methylphenyl)-3-(4-methoxyphenyl)-1H-pyrrol-1-yl)-2-chlorobenzoicacid

Synthesis:

Scheme 1, R=3-chloro-4-carboxyphenyl

Characterization Data:

¹H NMR (DMSO-d₆ 400 MHz TMS): δ 13.42 (brs, 1H), 7.97 (s, 1H), 7.71-7.66(m, 3H), 7.36 (m, 2H), 7.26 (d, J=7.6 Hz, 2H), 7.03 (m, 3H), 6.78 (d,J=8.4 Hz, 2H), 6.65 (d, J=2.8 Hz, 1H), 3.68 (s, 3H), 1.86 (s, 3H); MS(ESI): m/z 483.2 [M+23]⁺.

Example 214-(3-(4-methoxyphenyl)-1-(2-(trifluoromethylsulfonamido)ethyl)-1H-pyrrol-2-yl)-3-methylbenzamide

Synthesis:

Scheme 6

Characterization Data:

¹H NMR (CD₃OD 400 MHz TMS): δ 7.84-7.79 (m, 2H), 7.41 (d, J=7.6 Hz, 1H),7.00 (d, J=8.8 Hz, 2H), 6.92 (d, J=2.8 Hz, 1H), 6.70 (d, J=8.8 Hz, 2H),6.44 (d, J=2.8 Hz, 1H), 3.98-3.92 (m, 1H), 3.81-3.72 (m, 4H), 3.24 (t,J=6.4 Hz, 2H), 2.03 (s, 3H); MS (ESI): m/z 482.1 [M+1]⁺.

Example 224-(3-(4-methoxyphenyl)-1-(4-sulfamoylphenyl)-1H-pyrrol-2-yl)-3-methylbenzamide

Synthesis:

Scheme 1, R=4-sulfamoylphenyl

Characterization Data:

¹H NMR (CD₃OD 400 MHz TMS): δ7.78 (d, J=8.8 Hz, 2H), 7.67 (s, 1H), 7.61(d, J=8.0 Hz, 1H), 7.23-7.19 (m, 4H), 7.04 (d, J=8.8 Hz, 2H), 6.74 (d,J=8.8 Hz, 2H), 6.62 (d, J=4.4 Hz, 1H), 3.05 (s, 3H), 1.94 (s, 3H); MS(ESI) m/z: [M+23]⁺=484.0.

Example 234-(1-(3-fluoro-4-hydroxyphenyl)-3-(4-methoxyphenyl)-1H-pyrrol-2-yl)-3-methylbenzamide

Synthesis:

Scheme 1, R=3-fluoro-4-hydroxyphenyl, Step 1 uses 4-amino-2-fluorophenol(2A-1) described in Scheme 2A

Characterization Data:

¹H NMR (CD₃OD 400 MHz TMS): δ7.67 (s, 1H), 7.61 (d, J=8.0 Hz, 1H), 7.20(d, J=7.6 Hz, 1H), 7.04-7.02 (m, 3H), 6.82-6.71 (m, 5H), 6.53 (d, J=2.8Hz, 1H), 3.73 (s, 3H), 1.96 (s, 3H); MS (ESI) m/z: [M+1]⁺=417.1.

Example 245-(2-(4-carbamoyl-2-methylphenyl)-3-(4-methoxyphenyl)-1H-pyrrol-1-yl)picolinicacid

Synthesis:

Scheme 1, R=6-carboxypyridin-3-yl

Characterization Data:

¹H NMR (CD₃OD 400 MHz TMS): δ8.36 (s, 1H), 8.09 (d, J=8.4 Hz, 1H),7.72-7.70 (m, 2H), 7.66 (d, J=8.0 Hz, 1H), 7.30 (d, J=2.8 Hz, 1H), 7.24(d, J=8.0 Hz, 1H), 7.07 (d, J=8.8 Hz, 2H), 6.77 (d, J=8.8 Hz, 2H), 6.71(d, J=2.8 Hz, 1H), 3.75 (s, 3H), 1.97 (s, 3H); MS (ESI) m/z:[M+1]⁺=428.2.

Example 254-(1-(3,5-difluoro-4-hydroxyphenyl)-3-(4-methoxyphenyl)-1H-pyrrol-2-yl)-3-methylbenzamide

Synthesis:

Scheme 1, R=3,5-difluoro-4-hydroxyphenyl

Characterization Data:

¹H NMR (CDCl₃ 400 MHz TMS): δ7.59 (s, 1H), 7.47-7.49 (d, J=6.8 Hz, 1H),7.14-7.16 (d, J=8.0 Hz, 1H), 6.97-6.99 (d, J=8.8 Hz, 2H), 6.90-6.91 (d,1H), 6.68-6.71 (d, J=8.8 Hz, 2H), 6.56-6.58 (d, J=7.6 Hz, 2H), 6.51-6.52(d, 1H), 3.72 (s, 3H), 1.91 (s, 3H); MS (ESI): m/z 435.0 [M+1]⁺.

Example 264-(1-(5-hydroxypyridin-2-yl)-3-(4-methoxyphenyl)-1H-pyrrol-2-yl)-3-methylbenzamide

Synthesis:

Scheme 1, R=5-hydroxypyridin-2-yl, with modification of Step 1, where 1equivalent of AcOH was used instead of PPTS, and the reaction was heatedat 80° C. for 3 days. Purification by prep TLC.

Characterization Data:

¹H NMR (CD₃OD 400 MHz TMS): δ7.80 (d, J=3.2 Hz, 1H), 7.56 (s, 1H), 7.50(d, J=8.0 Hz, 1H), 7.14 (d, J=2.8 Hz, 1H), 7.08 (d, J=8.0 Hz, 1H),6.94-6.89 (m, 3H), 6.62 (d, J=8.8 Hz, 2H), 6.52 (d, J=8.8 Hz, 2H), 6.44(d, J=2.8 Hz, 1H), 3.62 (s, 3H), 1.84 (s, 3H); MS (ESI) m/z:[M+1]⁺=400.1.

Example 275-(2-(4-carbamoyl-2-methylphenyl)-3-(4-methoxyphenyl)-1H-pyrrol-1-yl)-2-hydroxybenzoicacid

Synthesis:

Scheme 1, R=2-hydroxybenzoic acid-4-yl, with modification of Step 1,where 1 equivalent of AcOH was used instead of PPTS.

Characterization Data:

¹H NMR (DMSO-d₆ 400 MHz TMS): δ7.92 (br, 1H), 7.64 (s, 1H), 7.60 (d,J=8.0 Hz, 1H), 7.46 (d, J=2.4 Hz, 1H), 7.33 (br, 1H), 7.20-7.17 (m, 2H),7.13 (d, J=3.2 Hz, 1H), 6.98 (d, J=8.8 Hz, 2H), 6.83 (d, J=8.8 Hz, 1H),6.74 (d, J=8.4 Hz, 2H), 6.54 (d, J=2.8 Hz, 1H), 3.65 (s, 3H), 1.85 (s,3H); MS (ESI) m/z: [M+1]⁺=443.1.

Example 286-(2-(4-carbamoyl-2-methylphenyl)-3-(4-methoxyphenyl)-1H-pyrrol-1-yl)nicotinicacid

Synthesis:

Scheme 1, R=5-carboxypyridin-2-yl, with modification of Step 1, whereAcOH was used as solvent instead of EtOH. The reaction was heated at130° C. for 4 hours. The mixture was concentrated in vacuo, and thecrude product was purified by reverse-phase preparatory HPLC (26-53%acetonitrile+0.1% trifluoroacetic acid in water+0.1% trifluoroaceticacid, over 15 min.).

Characterization Data:

¹H NMR (DMSO-d₆ 400 MHz TMS): δ 8.78 (s, 1H), 7.98 (s, 1H), 7.72 (s,1H), 7.66 (d, J=8.0, 1H), 7.56 (s, 1H), 7.37 (s, 1H), 7.21 (d, J=7.6,1H), 7.03 (d, J=8.4, 2H), 6.78 (d, J=8.8, 2H), 6.70 (d, J=6.8, 1H), 6.63(d, J=2.8, 1H), 3.69 (s, 3H), 1.86 (s, 1H); MS (ESI): m/z 428.2 [M+1]⁺.

Example 294-(1-(3-fluoro-4-hydroxyphenyl)-3-(4-(2-methyl-1H-imidazol-1-yl)phenyl)-1H-pyrrol-2-yl)-3-methylbenzamide

Synthesis:

Scheme 2A

Characterization Data:

¹H NMR (CD₃OD, 400 MHz, TMS): δ 7.63 (s, 1H), 7.59 (d, J=8.4 Hz, 1H),7.55 (d, J=2.0 Hz, 1H), 7.50 (d, J=2.0 Hz, 1H), 7.33-7.27 (m, 4H), 7.22(d, J=8.0 Hz, 1H), 7.04 (d, J=2.8 Hz, 1H), 6.88 (d, J=12.0 Hz, 1H),6.75-6.68 (m, 2H), 6.64 (d, J=2.8 Hz, 1H), 2.48 (s, 3H), 1.93 (s, 3H);MS (ESI): m/z 467.2 [M+1]⁺.

Example 304-(1-(3,5-difluoro-4-hydroxyphenyl)-3-(4-(2-methyl-1H-imidazol-1-yl)phenyl)-1H-pyrrol-2-yl)-3-methylbenzamide

Synthesis:

Scheme 2, R1=3,5-difluoro-4-hydroxyphenyl, R2=2-methyl-1H-imidazol-1-yl

Characterization Data:

¹H NMR (CD₃OD, 400 MHz, TMS): δ 7.75 (s, 1H), 7.71 (d, J=8.0 Hz, 1H),7.62 (d, J=2.0 Hz, 1H), 7.56 (d, J=2.0 Hz, 1H), 7.42-7.36 (m, 4H), 7.33(d, J=8.0 Hz, 1H), 7.16 (d, J=3.2 Hz, 1H), 6.77-6.74 (m, 3H), 2.56 (s,3H), 2.02 (s, 3H); MS (ESI): m/z 485.3 [M+1]⁺.

Example 31 General and Specific Methods of Preparing Novel GSNOR PyrroleInhibitors

This example describes schemes for preparing the GSNOR inhibitorsdepicted in Examples 1-30. The synthesis of 4 common intermediates,Intermediates A-D, are first described followed by the syntheticschemes. Some schemes are specific to a particular compound, whileothers are general schemes that include an exemplary method forpreparing a representative compound.

Synthesis of Common Intermediate A,4-(2-(4-methoxyphenyl)-4-oxobutanoyl)-3-methylbenzonitrile

Synthesis of A-2, (methyl 4-bromo-2-methylbenzoate)

To a solution of compound 1 (79.45 g, 369.5 mmol) in methanol (450 mL)was added thionyl chloride (53.6 mL, 739 mmol), and the mixture wasstirred at 70° C. for 6 h. The reaction mixture was concentrated toafford A-2 (86.37 g, yield 100%). ¹H NMR (CDCl₃ 300 MHz, TMS): δ 7.77(d, J=8.4 Hz, 1H), 7.40-7.34 (m, 2H), 3.87 (s, 3H), 2.56 (s, 3H).

Synthesis of A-3, (methyl 4-cyano-2-methylbenzoate)

A mixture of A-2 (methyl 4-bromo-2-methylbenzoate) (84.65 g, 369.5 mmol)and cuprous cyanide (45 g, 502.5 mmol) in dimethylformamide (60 mL) wasrefluxed for 8 h. After being cooled down to room temperature, thereaction mixture was diluted with water (400 mL), and the resultingmixture was filtrated. The filter cake was washed with ethyl acetate(200 mL×3). The filtrate was washed with water (400 mL), and the aqueouslayer was extracted with ethyl acetate (200 mL×2). The combined organiclayer was washed with brine (400 mL), dried over anhydrous sodiumsulfate (40 g), and evaporated. The residue was purified by silica gelcolumn chromatography (petroleum ether/ethyl acetate=15:1) to affordA-3, (methyl 4-cyano-2-methylbenzoate) (38 g, yield 58.7%) as a yellowsolid. ¹H NMR (CDCl₃ 400 MHz): δ 8.05 (d, J=39.6 Hz, 1H), 7.56 (d, J=8.0Hz, 2H), 3.96 (s, 3H), 2.65 (s, 3H).

Synthesis of A-4, (4-cyano-2-methylbenzoic acid)

To a solution of A-3 (methyl 4-cyano-2-methylbenzoate) (38 g, 0.217 mol)in ethanol (450 mL) was added aqueous sodium hydroxide solution (150 mL,2.9 M), and the mixture was stirred at room temperature for 12 h. Mostof ethanol was removed under reduced pressure, and the residue wasdiluted with water (300 mL). The aqueous layer was washed with ethylether (200 mL×2), and the aqueous layer was acidified with hydrochloricacid (450 mL, 1 M) under 0° C. to pH<6. The mixture was extracted withethyl acetate (400 mL×3), and the organic layer was washed with brine,dried, and evaporated to give A-4 (4-cyano-2-methylbenzoic acid) (40 g,yield 100%) as a yellow solid.

Synthesis of A-5, (4-cyano-2-methylbenzoyl chloride)

To a solution of A-4 (4-cyano-2-methylbenzoic acid) (20 g, 0.124 mol) intoluene (200 mL) was added thionyl chloride (18 mL, 0.248 mol), andcatalytic amount of dimethylformamide (0.5 mL). The resultant mixturewas stirred at 70° C. for 14 h, and concentrated under reduced pressureto give A-5 (4-cyano-2-methylbenzoyl chloride) (24 g, yield 100%), whichwas used in the next step without further purification.

Synthesis of A-6, (ethyl3-(4-cyano-2-methylphenyl)-2-(4-methoxyphenyl)-3-oxopropanoate)

To a solution of (4-methoxy-phenyl)-acetic acid ethyl ester (25.96 g,133.7 mmol) in anhydrous tetrahydrofuran (200 mL) was added lithiumhexamethyldisilazide (200 mL, 1 M) at −78° C. under nitrogen. Afterbeing stirred for 15 min., a solution of A-5 (4-cyano-2-methylbenzoylchloride) (24 g, 133.6 mmol) in anhydrous tetrahydrofuran (150 mL) wasadded dropwise, and the resulting mixture was stirred at roomtemperature overnight for 16 h. The reaction mixture was quenched by theaddition of saturated aqueous ammonium chloride solution (200 mL), andthe resultant mixture was extracted with ethyl acetate (100 mL×3). Thecombined organic layers were dried over anhydrous sodium sulfate (20 g),filtered, and evaporated. The residue was purified on silica gel column(petroleum ether/ethyl acetate=25:1) to give A-6 (ethyl3-(4-cyano-2-methylphenyl)-2-(4-methoxyphenyl)-3-oxopropanoate) (40 g,yield 88.7%).

Synthesis of A-7, (4-(2-(4-methoxyphenyl)acetyl)-3-methylbenzonitrile)

To a mixture of ethyl3-(4-cyano-2-methylphenyl)-2-(4-methoxyphenyl)-3-oxopropanoate (A-6) (40g, 0.1186 mol) in dimethyl sulfoxide (75 mL) was added catalytic amountof brine (2.5 mL), and the reaction mixture was heated at 150° C. for2.5 h. When TLC showed the starting material was consumed, the reactionmixture was cooled down to room temperature, and partitioned betweenwater (150 mL) and ethyl acetate (150 mL). The aqueous layer wasextracted with ethyl acetate (100 mL×2). The combined organic layer waswashed with brine (200 mL), dried over anhydrous sodium sulfate (15 g),evaporated, and purified by chromatography on silica gel (petroleumether/ethyl acetate=25:1) to give4-(2-(4-methoxyphenyl)acetyl)-3-methylbenzonitrile (A-7) (19 g, yield60.4%).

Synthesis of A-8,(4-(2-(4-methoxyphenyl)pent-4-enoyl)-3-methylbenzonitrile)

To a solution of A-7(4-(2-(4-methoxyphenyl)acetyl)-3-methylbenzonitrile) (19 g, 71.6 mmol)in anhydrous tetrahydrofuran (200 mL) was added dropwise lithiumhexamethyldisilazide (LiHMDS) (86 mL, 1 mol/L in THF) at −78° C. undernitrogen, and the reaction mixture was stirred at −78° C. for 30 min. Asolution of 3-bromo-propene (10.4 g, 86 mmol) in anhydroustetrahydrofuran (70 mL) was added dropwise into the mixture, and theresulting mixture was warmed to room temperature and stirred overnight.The reaction was quenched by the addition of saturated aqueous ammoniumchloride solution (200 mL), and extracted with ethyl acetate (100 mL×3).The combined organic layers were washed with brine (200 mL), dried overanhydrous sodium sulfate (10 g), evaporated, and purified on silica gelcolumn (petroleum ether/ethyl acetate=25:1) to give A-8(4-(2-(4-methoxyphenyl)pent-4-enoyl)-3-methylbenzonitrile) (7.7 g, yield35.2%) as an oil. ¹H NMR (CDCl₃ 400 MHZ): δ 7.36 (m, 3H), 7.01 (d, J=8.4Hz, 2H), 6.74 (d, J=8.4 Hz, 2H), 5.72-5.62 (m, 1H), 5.04-4.93 (m, 2H),4.20-4.17 (m, 1H), 3.69 (s, 3H), 2.92-2.85 (m, 1H), 2.52-2.40 (m, 1H),2.16 (s, 3H).

Synthesis of Intermediate A,(4-(2-(4-methoxyphenyl)-4-oxobutanoyl)-3-methylbenzonitrile)

To a solution of A-8(4-(2-(4-methoxyphenyl)pent-4-enoyl)-3-methylbenzonitrile) (9.1 g, 29.8mmol) in dichloromethane (400 mL) at −78° C. was bubbled with ozone in40 min. Dimethyl sulfide (20 mL) was slowly added into the solution at−78° C. After the addition, the resulting mixture was stirred at roomtemperature overnight, poured into water (150 mL), and extracted withdichloromethane (150 mL×2). The organic layer was washed with brine (150mL), dried over anhydrous sodium sulfate (15 g), evaporated, andpurified by silica gel (petroleum ether/ethyl acetate=5:1) to giveIntermediate A,4-(2-(4-methoxyphenyl)-4-oxobutanoyl)-3-methylbenzonitrile (3.0 g, yield32.8%) as a yellow solid. ¹H NMR (DMSO-d₆ 400 MHZ): δ 9.72 (s, 1H), 7.94(d, J=8.0 Hz, 1H), 7.77 (d, J=8.0 Hz, 1H), 7.69 (s, 1H), 7.12 (d, J=8.8Hz, 2H), 6.84 (d, J=8.4 Hz, 2H), 5.00-4.96 (m, 1H), 3.68 (s, 3H),3.49-3.42 (m, 1H), 2.91-2.85 (m, 1H), 2.11 (s, 3H).

Synthesis of Common Intermediate B,3-(3-(4-bromophenyl)-2-(4-cyano-2-methylphenyl)-1H-pyrrol-1-yl)propanoicacid

Synthesis of B-1, (ethyl2-(4-bromophenyl)-3-(4-cyano-2-methylphenyl)-3-oxopropanoate)

Followed same procedure as described the synthesis of Intermediate A,step 5 (transformation of A-5 to A-6) using ethyl2-(4-bromophenyl)acetate.

Synthesis of B-2, (4-(2-(4-bromophenyl)acetyl)-3-methylbenzonitrile)

To a solution of compound B-1 (8.0 g, 20.7 mmol) in DMSO (50 mL) wasadded catalytic amount of brine (0.3 mL), and the reaction mixture washeated at 165° C. for 2 h. When TLC showed the starting material wasconsumed, the reaction mixture was cooled down to room temperature, andpartitioned between water (150 mL) and ethyl acetate (150 mL). Theaqueous layer was extracted with ethyl acetate (50 mL×3). The combinedorganic layer was washed with brine, dried over sodium sulfate, andevaporated to give compound B-2,(4-(2-(4-bromophenyl)acetyl)-3-methylbenzonitrile) (4.5 g, yield: 71%)as a solid.

Synthesis of B-3,4-(2-(4-bromophenyl)pent-4-enoyl)-3-methylbenzonitrile

Followed same procedure as described the synthesis of Intermediate A,step 7 (transformation of A-7 to A-8). Purification of the crude wasaccomplished via silica gel column (eluting with 10% ethyl acetate inpetroleum ether) to give B-3,4-(2-(4-bromophenyl)pent-4-enoyl)-3-methylbenzonitrile (yield: 41%) asoil.

Synthesis of Intermediate B,4-(2-(4-bromophenyl)-4-oxobutanoyl)-3-methylbenzonitrile

A solution of compound B-3 (2.0 g, 7.6 mmol) in DCM (100 mL) was bubbledwith O₃ at −78° C. for 1 h. After the reaction mixture was warmed toroom temperature, it was quenched by the addition of SMe₂ (20 mL).Volatiles were evaporated and the residue was purified on silica gelcolumn (petroleum ether:ethyl acetate=3:1) to give Intermediate B,4-(2-(4-bromophenyl)-4-oxobutanoyl)-3-methylbenzonitrile (700 mg, yield34.8%) as an oil.

Synthesis of Intermediate C, ethyl2-(4-cyano-2-methylbenzoyl)-4-oxobutanoate

Synthesis of ethyl 3-(4-cyano-2-methylphenyl)-3-oxopropanoate, (C-1)

To a solution of diisopropylamine (7.57 g, 75 mmol) in anhydrous THF(150 mL) was added n-BuLi (30 mL, 75 mmol, 2.5 M in hexane) at −78° C.under nitrogen, after being stirred for 15 min., a solution of ethylacetate (6.33 g, 72 mmol) in anhydrous THF (20 mL) was added dropwise.The resulting mixture was stirred for an additional one hour, and asolution of compound A-5 (see synthesis of Intermediate A) in THF (50mL) was added dropwise. After the addition, the resulting mixture wasstirred at room temperature overnight. The reaction mixture was quenchedby saturated aqueous NH₄Cl solution (200 mL), and the resultant mixturewas extracted with ethyl acetate (100 mL×3). The combined organic layerswere dried over Na₂SO₄, filtered, and evaporated under vacuum. Theresidue was purified on silica gel column (eluting with 5-8% ethylacetate in petroleum ether) to give compound C-1 (4.1 g, yield: 30%).

Synthesis of ethyl 2-(4-cyano-2-methylbenzoyl)pent-4-enoate, (C-2)

To a solution of compound C-1 (4.1 g, 17.7 mmol) in anhydrous THF (60mL) at 0° C. under nitrogen was added NaH (60% in mineral, 1.0 g, 25mmol) in portions, after the addition, the reaction mixture was stirredat 0° C. for 30 min. A solution of 3-bromo-propene (2.68 mg, 22.2 mmol)in anhydrous THF (20 mL) was added dropwise into the mixture, and theresulting mixture was refluxed overnight. After being cooled down toroom temperature, the reaction mixture was quenched by the addition ofsaturated ammonium chloride solution (100 mL), and the resulting mixturewas extracted with ethyl acetate (50 mL×3). The combined organic layerwas washed with brine, dried over sodium sulfate, and evaporated. Theresidue was purified on silica gel column (eluting with 10% ethylacetate in petroleum ether) to give compound C-2 (2.8 g, yield: 58%) asan oil.

Synthesis of ethyl 2-(4-cyano-2-methylbenzoyl)-4-oxobutanoate,(Intermediate C)

A solution of compound C-2 (2.8 g, 10.3 mmol) in DCM (60 mL) was bubbledwith O₃ for 1 h at −78° C., after the reaction mixture was warmed toroom temperature, it was quenched by the addition of SMe₂ (5 mL).Volatiles were evaporated, and the residue was purified on silica gelcolumn (eluting with 30% ethyl acetate in petroleum ether) to giveIntermediate C (1.5 g, yield: 53%) as an oil.

Synthesis of Intermediate D, ethyl2-(4-bromo-2-methylbenzoyl)-4-oxobutanoate

Synthesis of 4-bromo-N-methoxy-N,2-dimethylbenzamide, (D-2)

To the solution of compound 4-bromo-2-methylbenzoic acid D-1 (50 g, 0.23mol) in DCM (500 mL) was added EDCI (48 g, 0.25 mol), HOBt (34.1 g, 0.25mol), NMM (50.5 g, 0.50 mol) and N,O-dimethylhydroxylamine hydrochloride(24.3 g, 0.25 mol). The reaction mixture was stirred at room temperatureovernight, diluted with DCM, washed with H₂O (300 mL), 1 N HCl (300 mL),and saturated sodium bicarbonate solution (300 mL), dried over sodiumsulfate, and concentrated to give the desired product D-2 (55 g, yield91.6%) as yellow oil.

Synthesis of ethyl 3-(4-bromo-2-methylphenyl)-3-oxopropanoate, (D-3)

Followed similar procedure to that described in the synthesis of C-1, ofIntermediate C synthesis. The only deviation was that the reaction wasallowed to stir 4 hours at −78° C. after final addition before warmingto room temperature.

Synthesis of ethyl 2-(4-bromo-2-methylbenzoyl)pent-4-enoate, (D-4)

Followed the procedure described in the synthesis of C-2, ofIntermediate C synthesis.

Synthesis of ethyl 2-(4-bromo-2-methylbenzoyl)-4-oxobutanoate,Intermediate D

Followed the procedure described in the final step of the synthesis ofIntermediate C.

Representative procedure for Scheme 1: Synthesis of3-(2-(4-carbamoyl-2-methylphenyl)-3-(4-methoxyphenyl)-1H-pyrrol-1-yl)butanoicacid (1-2, R=butanoic acid-3-yl) Synthesis of3-(2-(4-cyano-2-methylphenyl)-3-(4-methoxyphenyl)-1H-pyrrol-1-yl)butanoicacid (1-1, R=butanoic acid-3-yl)

A mixture of Intermediate A,(4-(2-(4-methoxyphenyl)-4-oxobutanoyl)-3-methylbenzonitrile), (100 mg,0.33 mmol), 3-aminobutanoic acid (67 mg, 0.65 mmol), and PPTS(Pyridinium p-toluenesulfonate) (8 mg, 0.033 mmol) in EtOH (10 mL) wasstirred at 60° C. for 2 hours. When LCMS indicated that Intermediate Awas consumed, the mixture was concentrated in vacuo to afford the crudedesired compound3-(2-(4-cyano-2-methylphenyl)-3-(4-methoxyphenyl)-1H-pyrrol-1-yl)butanoicacid (120 mg), which was used directly in the next step.

Synthesis of3-(2-(4-carbamoyl-2-methylphenyl)-3-(4-methoxyphenyl)-1H-pyrrol-1-yl)butanoicacid (1-2, R=butanoic acid-3-yl)

To a solution of the crude3-(2-(4-cyano-2-methylphenyl)-3-(4-methoxyphenyl)-1H-pyrrol-1-yl)butanoicacid (120 mg) in DMSO (5 mL) was added a solution NaOH (26 mg, 0.65mmol) in a mixture of water (0.5 mL) and H₂O₂ (0.33 mmol). After beingstirred at room temperature for 2 hours, the mixture was purified bypreparative HPLC to give 26 mg of3-(2-(4-carbamoyl-2-methylphenyl)-3-(4-methoxyphenyl)-1H-pyrrol-1-yl)butanoicacid (yield 20% in two steps) as a grey solid.

Representative procedure for Scheme 2: Synthesis of3-(3-(4-(1H-imidazol-1-yl)phenyl)-2-(4-carbamoyl-2-methylphenyl)-1H-pyrrol-1-yl)propanoicacid Synthesis of3-(3-(4-bromophenyl)-2-(4-cyano-2-methylphenyl)-1H-pyrrol-1-yl)propanoicacid (2-1, R1=propanoic acid-3-yl)

A mixture of Intermediate B (600 mg, 1.685 mmol), 3-amino-propionic acid(300 mg, 3.37 mmol), PPTS (41.4 mg, 0.168 mmol) in anhydrous ethanol (25mL) was stirred at room temperature overnight. The reaction mixture wasevaporated, and the residue was purified on silica gel column (elutingwith 1% to 2% methanol in dichloromethane) to give3-(3-(4-bromophenyl)-2-(4-cyano-2-methylphenyl)-1H-pyrrol-1-yl)propanoicacid (400 mg, yield 58.3%)

Synthesis of3-(3-(4-(1H-imidazol-1-yl)phenyl)-2-(4-cyano-2-methylphenyl)-1H-pyrrol-1-yl)propanoicacid (2-2, R1=propanoic acid-3-yl, R2=1H-imidazol-1-yl)

To a mixture of3-(3-(4-bromophenyl)-2-(4-cyano-2-methylphenyl)-1H-pyrrol-1-yl)propanoicacid (400 mg, 0.98 mmol) and imidazole (200 mg, 2.94 mol) in DMSO (5 mL)was added L-proline (33.8 mg, 0.294 mmol), CuI (111.7 mg, 0.588 mmol)and K2CO3 (270 mg, 1.96 mmol), then the resultant mixture was stirred at100° C. overnight. After being cooled down to room temperature, thereaction mixture was filtered, and the filter cake was washed with ethylacetate (10 mL×3). The combined filtrate was washed with saturatedNaHCO3 aqueous solution (30 mL), and the aqueous layer was extractedwith ethyl acetate (15 mL×3). The combined organic layers were washedwith brine, dried over MgSO4, concentrated. The residue was purified bysilica gel column (DCM: MeOH=30:1-20:1) to afford3-(3-(4-(1H-imidazol-1-yl)phenyl)-2-(4-cyano-2-methylphenyl)-1H-pyrrol-1-yl)propanoicacid (190 mg, yield 48.9%) as a solid.

Synthesis of3-(3-(4-(1H-imidazol-1-yl)phenyl)-2-(4-carbamoyl-2-methylphenyl)-1H-pyrrol-1-yl)propanoicacid (2-3, R1=propanoic acid-3-yl, R2=1H-imidazol-1-yl)

To a mixture of compound3-(3-(4-(1H-imidazol-1-yl)phenyl)-2-(4-cyano-2-methylphenyl)-1H-pyrrol-1-yl)propanoicacid (180 mg, 0.98 mmol) and sodium hydroxide (80 mg, 2.0 mol) in DMSO(5 mL) was added 0.1 mL of 30% hydrogen peroxide, and the mixture wasstirred at room temperature overnight. The reaction mixture wasneutralized by 1N HCl to PH=5-6, concentrated, and purified bypreparative HPLC to give3-(3-(4-(1H-imidazol-1-yl)phenyl)-2-(4-carbamoyl-2-methylphenyl)-1H-pyrrol-1-yl)propanoicacid (29 mg, yield 15.4%) as a solid.

Synthesis of 4-amino-2-fluorophenol (2A-1)

Under H₂, a mixture of 2-fluoro-4-nitrophenol (10 g, 63.7 mmol) and Pd/C(1 g) in methanol (100 mL) was stirred at room temperature for 5 hours.The mixture was filtered, and concentrated in vacuo to give the compound2A-1 (7 g, yield 86%).

Synthesis of4-(3-(4-bromophenyl)-1-(3-fluoro-4-hydroxyphenyl)-1H-pyrrol-2-yl)-3-methylbenzonitrile(2A-2)

Followed same procedure as described above in step 1 of Scheme 2.

Synthesis of4-(3-(4-bromophenyl)-1-(3-fluoro-4-(methoxymethoxy)phenyl)-1H-pyrrol-2-yl)-3-methylbenzonitrile(2A-3)

To a mixture of compound 2A-2 (400 mg, 0.89 mmol) in DMF (5 mL) wasadded NaH (54 mg, 1.34 mmol) at 0° C., and the mixture was stirred for 1hour. To the mixture, MOMCl (108 mg, 1.34 mmol) was added, and themixture was stirred for 1 hour. The mixture was quenched with waterslowly, and extracted with ethyl acetate (50 mL×3). The combined organiclayers were washed with brine, dried over Na₂SO₄, and concentrated togive the compound 2A-3 (370 mg, yield 84%).

Synthesis of4-(1-(3-fluoro-4-hydroxyphenyl)-3-(4-(2-methyl-1H-imidazol-1-yl)phenyl)-1H-pyrrol-2-yl)-3-methylbenzonitrile(2A-4)

Followed procedure described in step 2 of Scheme 2, where the reactionmixture was heated to 140° C. for 6 hours, workup was as described, andpurification was by silica gel column (PE: EA=5:1-1:1) to afford thecompound 2A-4 (160 mg, yield 50%).

Synthesis of4-(1-(3-fluoro-4-hydroxyphenyl)-3-(4-(2-methyl-1H-imidazol-1-yl)phenyl)-1H-pyrrol-2-yl)-3-methylbenzamide(2A-5)

Followed procedure described in step 3 of Scheme 2, except forpurification which was achieved by thin layer chromatography (CH₂Cl₂:MeOH=10:1) to give 2A-5 (16.3 mg, yield 15.4%).

Representative procedure for Scheme 3: Synthesis of3-(2-(4-carbamoyl-2-methylphenyl)-1-(4-chloro-2-methoxyphenyl)-1H-pyrrol-3-yl)propanoicacid (3-7, where R1=4-chloro-2-methoxyphenyl) Synthesis of ethyl1-(4-chloro-2-methoxyphenyl)-2-(4-cyano-2-methylphenyl)-1H-pyrrole-3-carboxylate(3-1, R1=4-chloro-2-methoxyphenyl)

To a solution of Intermediate C (2.5 g, 9.2 mmol) and4-chloro-2-methoxyaniline (1.8 g, 9.2 mmol) in anhydrous EtOH (20 mL)was added PPTS (0.46 g, 0.18 mmol), and the reaction mixture was heatedat 50° C. for six hours. Followed work-up procedure described in Step 1,Scheme 1 to give the desired compound, which was used in the next stepwithout further purification.

Synthesis of ethyl2-(4-carbamoyl-2-methylphenyl)-1-(4-chloro-2-methoxyphenyl)-1H-pyrrole-3-carboxylate(3-2, R1=4-chloro-2-methoxyphenyl)

To a solution of ethyl1-(4-chloro-2-methoxyphenyl)-2-(4-cyano-2-methylphenyl)-1H-pyrrole-3-carboxylate(5.0 g, 12.7 mmol) in DMSO (10 mL) was added aqueous NaOH solution (2mol/L, 1.0 mL) and 30% aqueous solution of H₂O₂ (1.0 mL), the reactionmixture was stirred at room temperature for four hours. TLC (EtOAc:MeOH=10:1) indicated that the starting material was consumed. Saturatedaqueous Na₂SO₃ solution was added to quench the reaction, and theaqueous layer was extracted with EtOAc (20 mL×3). The combined organiclayer was washed with brine (20 mL), dried over Na₂SO₄, filtered,concentrated, and purified by column chromatograph to afford the titledcompound as a yellow oil (1.2 g, yield: 23.1%). MS (ESI): m/z 413.1[M+1]⁺.

Synthesis of4-(1-(4-chloro-2-methoxyphenyl)-3-(hydroxymethyl)-1H-pyrrol-2-yl)-3-methylbenzamide(3-3, R1=4-chloro-2-methoxyphenyl)

A solution of ethyl2-(4-carbamoyl-2-methylphenyl)-1-(4-chloro-2-methoxyphenyl)-1H-pyrrole-3-carboxylate(1.2 g, 2.9 mmol) in anhydrous toluene (50 mL) was cooled to −78° C.,DIBAL-H (1.0 mol/L, 14.5 mL) was added dropwise, and the reactionmixture was stirred at this temperature for two hours. TLC (EtOAc:MeOH=10:1) indicated that the starting material was consumed. Saturatedaqueous NH₄Cl solution was added to quench the reaction, and the mixturewas filtered. The filtrate was extracted with EtOAc (20 mL×3), and thecombined organic layer was washed with brine (20 mL), dried over Na₂SO₄,filtered, concentrated, and purified by column chromatography to givethe titled compound (430 mg, yield: 40.2%). ¹H NMR (CD₃OD 300 MHz TMS):δ 7.63 (s, 1H), 7.55 (m, 1H), 7.21 (d, J=7.8 Hz, 1H), 7.03 (d, J=8.1 Hz,1H), 6.95 (d, J=2.1 Hz, 1H), 6.82-6.87 (m, 2H), 6.43 (d, J=3.0 Hz, 1H),4.38 (d, J=11.7 Hz, 1H), 4.27 (d, J=11.7 Hz, 1H), 3.60 (s, 3H), 2.10 (s,3H).

Synthesis of4-(1-(4-chloro-2-methoxyphenyl)-3-formyl-1H-pyrrol-2-yl)-3-methylbenzamide,(3-4, R1=4-chloro-2-methoxyphenyl)

To a solution of4-(1-(4-chloro-2-methoxyphenyl)-3-(hydroxymethyl)-1H-pyrrol-2-yl)-3-methylbenzamide(430 mg, 1.2 mmol) in CH₂Cl₂ (10 mL) was added MnO₂ (562 mg, 6.5 mmol),the reaction mixture was refluxed for two days. TLC (EtOAc: MeOH=10:1)indicated that the starting materials were consumed. The reactionmixture was filtered, and the filtrate was concentrated to give thedesired compound (320 mg, yield: 75.1%), which was used in the next stepwithout further purification.

Synthesis of (E)-ethyl3-(2-(4-carbamoyl-2-methylphenyl)-1-(4-chloro-2-methoxyphenyl)-1H-pyrrol-3-yl)acrylate,(3-5, R1=4-chloro-2-methoxyphenyl)

To a solution of4-(1-(4-chloro-2-methoxyphenyl)-3-formyl-1H-pyrrol-2-yl)-3-methylbenzamide(320 mg, 0.87 mmol) in anhydrous toluene (10 mL) was added compoundPh₃P═CHCO₂Et (440 mg, 1.3 mmol), and the reaction mixture was refluxedovernight. The solvent was removed in vacuo, and the residue waspurified by column chromatography (eluting with ethyl acetate inpetroleum ether) to give the crude compound (E)-ethyl3-(2-(4-carbamoyl-2-methylphenyl)-1-(4-chloro-2-methoxyphenyl)-1H-pyrrol-3-yl)acrylate(610 mg), which contents some of Ph₃PO. ¹H NMR (CD₃OD 400 MHz TMS): δ7.54-7.72 (m, 2H), 7.25 (d, J=15.6 Hz, 1H), 7.12-7.20 (m, 2H), 7.00 (s,1H), 6.87-6.92 (m, 2H), 6.70 (d, J=3.2 Hz, 1H), 6.25 (d, J=15.6 Hz, 1H),4.15 (q, J=7.2 Hz, 2H), 3.63 (s, 3H), 2.12 (s, 3H), 1.25 (t, J=7.2 Hz,3H).

Synthesis of ethyl3-(2-(4-carbamoyl-2-methylphenyl)-1-(4-chloro-2-methoxyphenyl)-1H-pyrrol-3-yl)propanoate,(3-6, R1=4-chloro-2-methoxyphenyl)

To a solution of (E)-ethyl3-(2-(4-carbamoyl-2-methylphenyl)-1-(4-chloro-2-methoxyphenyl)-1H-pyrrol-3-yl)acrylate(610 mg, 1.4 mmol) in THF (10 mL) was added Raney Ni (100 mg), and thereaction mixture was hydrogenated at room temperature under 1 atm ofhydrogen for two hours. The reaction mixture was filtered, and thefiltrate was concentrated to give the title compound (610 mg), which wasused in the next step without further purification.

Synthesis of3-(2-(4-carbamoyl-2-methylphenyl)-1-(4-chloro-2-methoxyphenyl)-1H-pyrrol-3-yl)propanoicacid, (3-7, R1=4-chloro-2-methoxyphenyl)

To a solution of ethyl3-(2-(4-carbamoyl-2-methylphenyl)-1-(4-chloro-2-methoxyphenyl)-1H-pyrrol-3-yl)propanoate(580 mg, 1.3 mmol) in MeOH (5 mL) was added aqueous LiOH solution (2mol/L, 5 mL), and the reaction mixture was stirred at room temperaturefor three hours. TLC (PE: EtOAc=1:2) showed that the starting materialwas consumed. The aqueous layer was washed with EtOAc (10 mL), andacidified by 0.5 N HCl to pH=2. The aqueous layer was extracted with amixture of EtOAc and MeOH (v/v=10:1, 20 mL×3), and the combined organiclayer was washed with brine (20 mL), dried over Na₂SO₄, filtered, andconcentrated. The residue was purified by column chromatography andpreparative HPLC to afford the titled compound as an off-white solid(68.3 mg, yield: 12.6%).

Representative procedure for Scheme 4: Synthesis of3-(2-(4-carbamoyl-2-methylphenyl)-1-(4-methoxyphenyl)-1H-pyrrol-3-yl)propanoicacid (4-2, R1=4-methoxyphenyl) Synthesis of ethyl2-(4-bromo-2-methylphenyl)-1-(4-methoxyphenyl)-1H-pyrrole-3-carboxylate(4-1, R1=4-methoxyphenyl)

A mixture of Intermediate D (5 g, 15.3 mmol), 4-methoxy-phenylamine (1.9mg, 15.3 mmol), PPTS (140 mg, 0.57 mmol) in anhydrous ethanol (100 mL)was stirred at room temperature overnight. The reaction mixture wasevaporated, and purified by silica gel column (eluting with 20% ethylacetate in petroleum ether) to give the desired compound (1 g, yield15.7%) as a solid.

Synthesis of ethyl2-(4-carbamoyl-2-methylphenyl)-1-(4-methoxyphenyl)-1H-pyrrole-3-carboxylate(3-2, R1=4-methoxyphenyl)

To a mixture of ethyl2-(4-bromo-2-methylphenyl)-1-(4-methoxyphenyl)-1H-pyrrole-3-carboxylate(1 g, 2.4 mmol) in DMF (50 mL) was added CuCN (0.43 g, 4.8 mmol), andthe suspension solution was heated to 180° C. for 12 h. After beingcooled down to room temperature, the reaction mixture was poured intowater, and the mixture was extracted with ethyl acetate (50 mL×3). Thecombined organic layers were washed with brine, dried over Na₂SO₄, andconcentrated under reduced pressure. The residue was purified on silicagel column chromatography (eluting with 50% ethyl acetate in petroleumether) to give the title compound (600 mg, 66% yield) as a white solid.

Synthesis of ethyl2-(4-carbamoyl-2-methylphenyl)-1-(4-methoxyphenyl)-1H-pyrrole-3-carboxylate(3-5, R1=4-methoxyphenyl)

Transformation of ethyl2-(4-carbamoyl-2-methylphenyl)-1-(4-methoxyphenyl)-1H-pyrrole-3-carboxylate(3-2, R1=4-methoxyphenyl) to the desired ethyl2-(4-carbamoyl-2-methylphenyl)-1-(4-methoxyphenyl)-1H-pyrrole-3-carboxylate(3-5, R1=4-methoxyphenyl) followed the same 3 steps described in theexample synthesis for Scheme 3, (3-2→3-3→3-4→3-5).

Synthesis of3-(2-(4-carbamoyl-2-methylphenyl)-1-(4-methoxyphenyl)-1H-pyrrol-3-yl)propanoicacid (4-2, R1=4-methoxyphenyl)

To a mixture of ethyl2-(4-carbamoyl-2-methylphenyl)-1-(4-methoxyphenyl)-1H-pyrrole-3-carboxylate(240 mg, 0.4 mmol) in methanol (10 mL) was added 10% palladium onactivated carbon (50 mg), and the mixture was hydrogenated under 1 atmof hydrogen at room temperature over 2 h. The reaction mixture wasfiltrated, concentrated under reduced pressure, and dissolved in amixture of ethanol (10 mL) and 1N NaOH solution (5 mL). The resultingmixture was stirred at room temperature overnight, the organic solventwas evaporated, and the mixture was adjusted to pH=3 with 1N HCl. Theaqueous layer was extracted with EtOAc (10 mL×3), and the organic layerswere washed with brine, dried over sodium sulfate, concentrated underreduced pressure, and purified by preparative HPLC to give the titlecompound (75 mg, yield: 30% in two steps).

Representative procedure for Scheme 5: Synthesis of3-(1-(4-(1H-imidazol-1-yl)phenyl)-2-(4-carbamoyl-2-methylphenyl)-1H-pyrrol-3-yl)propanoicacid (5-4, R1=1H-imidazol-1-yl) Synthesis of (E)-ethyl3-(1-(4-bromophenyl)-2-(4-carbamoyl-2-methylphenyl)-1H-pyrrol-3-yl)acrylate(5-1)

Described in Scheme 3, compound 3-5, where R1=4-bromophenyl.

Synthesis of ethyl3-(1-(4-bromophenyl)-2-(4-carbamoyl-2-methylphenyl)-1H-pyrrol-3-yl)propanoate(5-2)

To an ice cooled solution of (E)-ethyl3-(1-(4-bromophenyl)-2-(4-carbamoyl-2-methylphenyl)-1H-pyrrol-3-yl)acrylate(400 mg, 0.88 mmol) in 95% ethanol (10 mL) were added BiCl₃ (554 mg,1.76 mmol) and portionwise NaBH₄ (134 mg, 3.52 mmol), then the resultantmixture was stirred at room temperature for 12 h. The reaction mixturewas filtered, and the filtrate was partitioned between water (50 mL) andethyl acetate (30 mL), the aqueous layer was extracted with ethylacetate (30 mL×2). The combined organic layers were washed with brine,dried over sodium sulfate, and evaporated. The residue was purified bypreparative TLC (eluting with 50% ethyl acetate in petroleum ether forthree times) to give ethyl3-(1-(4-bromophenyl)-2-(4-carbamoyl-2-methylphenyl)-1H-pyrrol-3-yl)propanoate(5-2) (100 mg, yield: 25%).

Synthesis of ethyl3-(1-(4-(1H-imidazol-1-yl)phenyl)-2-(4-carbamoyl-2-methylphenyl)-1H-pyrrol-3-yl)propanoate,(5-3, R1=1H-imidazol-1-yl)

A mixture of ethyl3-(1-(4-bromophenyl)-2-(4-carbamoyl-2-methylphenyl)-1H-pyrrol-3-yl)propanoate(50 mg, 0.11 mmol), imidazole (68 mg, 10 mmol), L-proline (13 mg, 0.11mmol), CuI (42 mg, 0.22 mmol), K₂CO₃ (76 mg, 0.55 mmol) and 4 Åmolecular sieve (100 mg) in DMSO (3 mL) was heated at 130° C. for 24 h.After being cooled down to room temperature, the reaction mixture wasfiltered, and the filter cake was washed with ethyl acetate (10 mL×3).The combined filtrate was washed with saturated NaHCO₃ aqueous solution(30 mL), and the aqueous layer was extracted with ethyl acetate (15mL×3). The combined organic layers were washed with brine, dried overMgSO₄, and concentrated. The residue was purified by preparative TLC(eluting with 7% methanol in dichloromethane) to give ethyl3-(1-(4-(1H-imidazol-1-yl)phenyl)-2-(4-carbamoyl-2-methylphenyl)-1H-pyrrol-3-yl)propanoate,(5-3, R1=1H-imidazol-1-yl) (25 mg, yield: 51%), which was used in thenext step without further purification.

Synthesis of3-(1-(4-(1H-imidazol-1-yl)phenyl)-2-(4-carbamoyl-2-methylphenyl)-1H-pyrrol-3-yl)propanoicacid, (5-4, R1=1H-imidazol-1-yl)

A mixture of ethyl3-(1-(4-(1H-imidazol-1-yl)phenyl)-2-(4-carbamoyl-2-methylphenyl)-1H-pyrrol-3-yl)propanoate(25 mg, 0.057 mmol) and LiOH monohydrate (42 mg, 1 mmol) in a mixture ofmethanol (1.5 mL) and water (0.5 mL), and the reaction mixture wasstirred at room temperature overnight. Methanol was removed underreduced pressure, and the residue was diluted with water (5 mL), and theaqueous layer was washed with ethyl acetate (5 mL×3). The aqueous layerwas adjusted to PH=6-7 with 1N HCl, and the resulting mixture wasextracted with a mixture of dichloromethane and isopropanol (3:1, 5mL×3). The combined organic layer was dried, and evaporated to give3-(1-(4-(1H-imidazol-1-yl)phenyl)-2-(4-carbamoyl-2-methylphenyl)-1H-pyrrol-3-yl)propanoicacid, (5-4, R1=1H-imidazol-1-yl) (12 mg, yield: 51%) as a yellow solid.

Synthesis of tert-butyl2-(2-(4-cyano-2-methylphenyl)-3-(4-methoxyphenyl)-1H-pyrrol-1-yl)ethylcarbamate(6-1)

Followed procedure described in Step 1 of Scheme 1.

Synthesis of4-(1-(2-aminoethyl)-3-(4-methoxyphenyl)-1H-pyrrol-2-yl)-3-methylbenzonitrile(6-2)

A solution of tert-butyl2-(2-(4-cyano-2-methylphenyl)-3-(4-methoxyphenyl)-1H-pyrrol-1-yl)ethylcarbamate(6-1) (110 mg, 0.255 mmol) in methanolic hydrochloride solution (2 M, 5mL) was stirred at room temperature for 1 hour. The mixture wasconcentrated in vacuo to give the title compound 6-2 (100 mg), which wasused directly in the next step.

Synthesis ofN-(2-(2-(4-cyano-2-methylphenyl)-3-(4-methoxyphenyl)-1H-pyrrol-1-yl)ethyl)-1,1,1-trifluoromethanesulfonamide(6-3)

To a solution of4-(1-(2-aminoethyl)-3-(4-methoxyphenyl)-1H-pyrrol-2-yl)-3-methylbenzonitrile(6-2) (100 mg, 0.3 mmol) in anhydrous DCM (5 mL) was added pyridine (48mg, 0.6 mmol), followed by addition of trifluoromethanesulfonicanhydride (85 mg, 0.6 mmol) at 0° C. The mixture was stirred at roomtemperature overnight. When TLC showed the starting material wasconsumed, the mixture was partitioned between water and ethyl acetate,and the aqueous layer was extracted with ethyl acetate. The combinedorganic layer was concentrated under reduced pressure to give the titlecompound 6-3 (120 mg, yield 86%) as brown oil.

Synthesis of4-(3-(4-methoxyphenyl)-1-(2-(trifluoromethylsulfonamido)ethyl)-1H-pyrrol-2-yl)-3-methylbenzamide(6-4)

Followed step 2 of Scheme 1 to give the desired compound4-(3-(4-methoxyphenyl)-1-(2-(trifluoromethylsulfonamido)ethyl)-1H-pyrrol-2-yl)-3-methylbenzamide(6-4).

Example 32 GSNOR Assays

Various compounds were tested in vitro for their ability to inhibitGSNOR activity. GSNOR inhibitor compounds in Examples 1-30 had an IC₅₀of about <100 μM. GSNOR inhibitor compounds Examples 1, 3, 5, 6, 9,13-17, 19-20, 23, 25-27, 29-30 had an IC₅₀ of about <5 μM. GSNORinhibitor compounds Examples 1, 5-6, 13-15, 17, 19, 23, 25, 27, 29-30had an IC₅₀ of about less than 1.0 μM. GSNOR expression and purificationis described in Biochemistry 2000, 39, 10720-10729.

GSNOR Fermentation:

Pre-cultures were grown from stabs of a GSNOR glycerol stock in 2XYTmedia containing 100 ug/ml ampicillin after an overnight incubation at37° C. Cells were then added to fresh 2XYT (4 L) containing ampicillinand grown to an OD (A₆₀₀) of 0.6-0.9 at 37° C. before induction. GSNORexpression was induced with 0.1% arabinose in an overnight incubation at20° C.

GSNOR Purification:

E. coli cell paste was lysed by nitrogen cavitation and the clarifiedlysate purified by Ni affinity chromatography on an AKTA FPLC (AmershamPharmacia). The column was eluted in 20 mM Tris pH 8.0/250 mM NaCl witha 0-500 mM imidazole gradient. Eluted GSNOR fractions containing theSmt-GSNOR fusion were digested overnight with Ulp-1 at 4° C. to removethe affinity tag then re-run on the Ni column under the same conditions.GSNOR was recovered in the flowthrough fraction and for crystallographyis further purified by Q-Sepharose and Heparin flowthroughchromatography in 20 mM Tris pH 8.0, 1 mM DTT, 10 uM ZnSO₄.

GSNOR Assay:

GSNO and Enzyme/NADH Solutions are made up fresh each day. The Solutionsare filtered and allowed to warm to room temperature. GSNO Solution: 100mM NaPO4 (pH 7.4), 0.480 mM GSNO. 396 μL of GSNO Solution is added to acuvette followed by 8 μL of test compound in DMSO (or DMSO only for fullreaction control) and mixed with the pipette tip. Compounds to be testedare made up at a stock concentration of 10 mM in 100% DMSO. 2 foldserial dilutions are done in 100% DMSO. 8 μL of each dilution are addedto an assay so that the final concentration of DMSO in the assay is 1%.The concentrations of compounds tested range from 100 to 0.003 μM.Enzyme/NADH Solution: 100 mM NaPO4 (pH 7.4), 0.600 mM NADH, 1.0 μg/mLGSNO Reductase. 396 μL of the Enzyme/NADH Solution is added to thecuvette to start the reaction. The cuvette is placed in the Cary 3EUV/Visible Spectrophotometer and the change in 340 nm absorbance/min at25° C. is recorded for 3 minutes. The assays are done in triplicate foreach compound concentration. IC₅₀'s for each compound are calculatedusing the standard curve analysis in the Enzyme Kinetics Module ofSigmaPlot.

Final assay conditions: 100 mM NaPO4, pH 7.4, 0.240 mM GSNO, 0.300 mMNADH, 0.5 μg/mL GSNO Reductase and 1% DMSO. Final volume: 800μL/cuvette.

Example 33 Efficacy of GSNORi in Experimental Asthma

Experimental Asthma Model:

A mouse model of ovalbumin (OVA)-induced asthma is used to screen GSNORinhibitors for efficacy against methacholine (MCh)-inducedbronchoconstriction/airway hyper-reactivity. This is a widely used andwell characterized model that presents with an acute, allergic asthmaphenotype with similarities to human asthma. Efficacy of GSNORinhibitors are assessed using a prophylactic protocol in which GSNORinhibitors are administered prior to challenge with MCh.Bronchoconstriction in response to challenge with increasing doses ofMCh is assessed using whole body plethysmography (P_(enh): Buxco). Theamount of eosinophil infiltrate into the bronchoaveolar lavage fluid(BALF) is also determined as a measure of lung inflammation. The effectof GSNOR inhibitors are compared to vehicles and to Combivent (inhaled;1H) as the positive control.

Materials and Methods

Allergen Sensitization and Challenge Protocol

OVA (500 μg/ml) in PBS is mixed with equal volumes of 10% (w/v) aluminumpotassium sulfate in distilled water and incubated for 60 min. at roomtemperature after adjustment to pH 6.5 using 10 N NaOH. Aftercentrifugation at 750×g for 5 min, the OVA/alum pellet is resuspended tothe original volume in distilled water. Mice receive an intraperitoneal(IP) injection of 100 μg OVA (0.2 mL of 500 μg/mL in normal saline)complexed with alum on day 0. Mice are anesthetized by IP injection of a0.2-mL mixture of ketamine and xylazine (0.44 and 6.3 mg/mL,respectively) in normal saline and are placed on a board in the supineposition. Two hundred fifty micrograms (100 μl of a 2.5 mg/ml) of OVA(on day 8) and 125 μg (50 μl of 2.5 mg/ml) OVA (on days 15, 18, and 21)are placed on the back of the tongue of each animal.

Pulmonary Function Testing (Penh)

In vivo airway responsiveness to methacholine is measured 24 h after thelast OVA challenge in conscious, freely moving, spontaneously breathingmice with whole body plethysmography using a Buxco chamber (Wilmington,N.C.). Mice are challenged with aerosolized saline or increasing dosesof methacholine (5, 20 and 50 mg/mL) generated by an ultrasonicnebulizer for 2 min. The degree of bronchoconstriction is expressed asenhanced pause (P_(enh)), a calculated dimensionless value, whichcorrelates with the measurement of airway resistance, impedance, andintrapleural pressure in the same mouse. P_(enh) readings are taken andaveraged for 4 min. after each nebulization challenge. P_(enh) iscalculated as follows: P_(enh)=[(T_(e)/T_(r)−1)×(PEF/PIF)], where T_(e)is expiration time, T_(r) is relaxation time, PEF is peak expiratoryflow, and PIF is peak inspiratory flow×0.67 coefficient. The time forthe box pressure to change from a maximum to a user-defined percentageof the maximum represents the relaxation time. The T_(r) measurementbegins at the maximum box pressure and ends at 40%.

Eosinophil Infiltrate in BALF

After measurement of airway hyper-reactivity, the mice are exsanguinatedby cardiac puncture, and then BALF is collected from either both lungsor from the right lung after tying off the left lung at the mainstembronchus. Total BALF cells are counted from a 0.05 mL aliquot, and theremaining fluid is centrifuged at 200×g for 10 min at 4° C. Cell pelletsare resuspended in saline containing 10% BSA with smears made on glassslides. Eosinophils are stained for 5 min. with 0.05% aqueous eosin and5% acetone in distilled water, rinsed with distilled water, andcounterstained with 0.07% methylene blue.

GSNOR Inhibitors and Controls

GSNOR inhibitors are reconstituted in phosphate buffered saline (PBS),pH 7.4, at concentrations ranging from 0.00005 to 3 mg/mL. GSNORinhibitors are administered to mice (10 mL/kg) as a single dose eitherintravenously (IV) or orally via gavage. Dosing is performed from 30min. to 24 h prior to MCh challenge. Effect of GSNOR inhibitors arecompared to PBS vehicle dosed in the same manner.

Combivent is used as the positive control in all studies. Combivent(Boehringer Ingelheim) is administered to the lung using the inhalerdevice supplied with the product, but adapted for administration tomice, using a pipet tip. Combivent is administered 48 h, 24 h, and 1 hprior to MCh challenge. Each puff (or dose) of Combivent provides a doseof 18 μg ipatropium bromide (IpBr) and 103 μg albuterol sulfate orapproximately 0.9 mg/kg IpBr and 5 mg/kg albuterol.

Statistical Analyses

Area under the curve values for P_(enh) across baseline, saline, andincreasing doses of MCh challenge are calculated using GraphPad Prism5.0 (San Diego, Calif.) and expressed as a percent of the respective (IVor orally administered) vehicle control. Statistical differences amongtreatment groups and the respective vehicle control group within eachstudy are calculated using one-way ANOVA, Dunnetts (JMP 8.0, SASInstitute, Cary, N.C.). A p value of <0.05 among the treatment groupsand the respective vehicle control group is considered significantlydifferent.

It will be apparent to those skilled in the art that variousmodifications and variations can be made in the methods and compositionsof the present invention without departing from the spirit or scope ofthe invention.

1. A method of treatment of chronic inflammatory disease comprising administering a therapeutically effective amount of a compound of formula I:

wherein R₁ is selected from the group consisting of R₅ and R₆; R₁′ is R₅ when R₁ is R₆, and R₆ when R₁ is R₅; R₂ is selected from the group consisting of hydrogen, halogen, hydroxyl, C₁-C₆ alkyl, C₃-C₆ cycloalkyl, cyano, nitro, trifluoromethyl, carbamoyl, C₁-C₆ alkylcarbamoyl, amino, C₁-C₆ alkylamino, C₁-C₆ dialkylamino, C₁-C₆ alkoxyl, and C₃-C₆ cycloalkoxyl; R₃ is selected from the group consisting of halogen, hydroxyl, carbamoyl, substituted carbamoyl, C₁-C₆ alkylcarbamoyl, sulfamoyl, C₁-C₆ alkylsulfamoyl, C₁-C₆ alkyl, C₃-C₆ cycloalkyl, cyano, nitro, amino, CF₃, carboxyl, ureido, sulfamoylamino, 2-amino-2-oxoethyl, C₁-C₆ alkylamino, C₁-C₆ dialkylamino, arylamino, heteroarylamino, C₁-C₆ alkoxyl, C₃-C₆ cycloalkoxyl, aryl, substituted aryl, heteroaryl, and substituted heteroaryl; R₄ is selected from the group consisting of hydrogen, hydroxyl, halogen, C₁-C₆ alkyl, C₃-C₆ cycloalkyl, cyano, nitro, carbamoyl, C₁-C₆ alkylcarbamoyl, sulfamoyl, C₁-C₆ alkyl sulfamoyl, amino, C₁-C₆ alkylamino, C₁-C₆ dialkylamino, C₁-C₆ alkoxyl, and C₃-C₆ cycloalkoxyl; R₅ is selected from the group consisting of aryl, substituted aryl, heteroaryl, and substituted heteroaryl; R₆ is selected from the group consisting of —CR₇R₈CR₉R₁₀ (CR₁₁R₁₂)_(n)R₁₃ and

R₇, R₈, R₉, R₁₀, R₁₁, and R₁₂ are each independently selected from the group consisting of hydrogen, hydroxyl, halogen, C₁-C₆ alkyl, C₃-C₆ cycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl and wherein R₉ and R₁₀ together form a C₃-C₆ cycloalkyl; R₁₃ is selected from the group consisting of COOH and NH₂SO₂CF₃; R₁₄ is selected from the group consisting of OH, COOH, and SO₂NH₂; R₁₅ and R₁₆ are independently selected from the group consisting of hydrogen, hydroxyl, carboxyl, NO₂, and halogen; n is 0 or 1; and X and Y are independently selected from the group consisting of C and N.
 2. The method of claim 1 wherein R₅ is selected from the group consisting of 4-chlorophenyl, 3-chlorophenyl, 4-chloro-2-methoxyphenyl, 4-bromophenyl, 3-bromophenyl, 4-fluorophenyl, 3-fluorophenyl, 4-hydroxyphenyl, 4-methoxyphenyl, 3-methoxyphenyl, 2-methoxyphenyl, 4-chlorothiophen-2-yl, 5-chlorothiophen-2-yl, 3-bromothiophen-2-yl, 4-bromothiophen-2-yl, 5-bromothiopheny-2-yl, 5-bromothiophen-3-yl,

wherein R₁₇ is selected from the group consisting of hydrogen, methyl, chloro, fluoro, hydroxy, methoxy, ethoxy, propoxy, carbamoyl, dimethylamino, amino, formamido, and trifluoromethyl; and R₁₈ is selected from the group consisting of hydrogen, methyl, and ethyl.
 3. The method of claim 2 wherein R₂ is C₁-C₆ alkyl, R₃ is carbamoyl, and R₄ is hydrogen.
 4. The method of claim 3 wherein R₅ is selected from the group consisting of 4-methoxyphenyl, 4-chloro-2-methoxyphenyl, 4-(1H-imidazol-1-yl)phenyl, and 4-(2-methyl-1H-imidazol-1-yl)phenyl.
 5. The method of claim 1 wherein the compound of formula I is selected from the group consisting of: 3-(2-(4-carbamoyl-2-methylphenyl)-3-(4-methoxyphenyl)-1H-pyrrol-1-yl)propanoic acid; 3-(2-(4-carbamoyl-2-methylphenyl)-3-(4-methoxyphenyl)-1H-pyrrol-1-yl)-3-phenylpropanoic acid; 3-(2-(4-carbamoyl-2-methylphenyl)-3-(4-methoxyphenyl)-1H-pyrrol-1-yl)-2-methylpropanoic acid; 4-(2-(4-carbamoyl-2-methylphenyl)-3-(4-methoxyphenyl)-1H-pyrrol-1-yl)butanoic acid; 3-(3-(4-(1H-imidazol-1-yl)phenyl)-2-(4-carbamoyl-2-methylphenyl)-1H-pyrrol-1-yl)propanoic acid; 3-(2-(4-carbamoyl-2-methylphenyl)-1-(4-methoxyphenyl)-1H-pyrrol-3-yl)propanoic acid; 3-(2-(4-carbamoyl-2-methylphenyl)-3-(4-methoxyphenyl)-1H-pyrrol-1-yl)butanoic acid; 1-((2-(4-carbamoyl-2-methylphenyl)-3-(4-methoxyphenyl)-1H-pyrrol-1-yl)methyl)cyclopropanecarboxylic acid; 3-(2-(4-carbamoyl-2-methylphenyl)-3-(4-methoxyphenyl)-1H-pyrrol-1-yl)-2,2-difluoropropanoic acid; 3-(2-(4-carbamoyl-2-methylphenyl)-3-(4-methoxyphenyl)-1H-pyrrol-1-yl)-2-hydroxypropanoic acid; 3-(2-(4-carbamoyl-2-methylphenyl)-3-(4-methoxyphenyl)-1H-pyrrol-1-yl)hexanoic acid; 3-(2-(4-carbamoyl-2-methylphenyl)-3-(4-methoxyphenyl)-1H-pyrrol-1-yl)-4-methylpentanoic acid; 3-(1-(4-(1H-imidazol-1-yl)phenyl)-2-(4-carbamoyl-2-methylphenyl)-1H-pyrrol-3-yl)propanoic acid; 3-(2-(4-carbamoyl-2-methylphenyl)-1-(4-chloro-2-methoxyphenyl)-1H-pyrrol-3-yl)propanoic acid; 4-(2-(4-carbamoyl-2-methylphenyl)-3-(4-methoxyphenyl)-1H-pyrrol-1-yl)-2-hydroxybenzoic acid; 4-(2-(4-carbamoyl-2-methylphenyl)-3-(4-methoxyphenyl)-1H-pyrrol-1-yl)-2-fluorobenzoic acid; 4-(1-(4-hydroxy-3-nitrophenyl)-3-(4-methoxyphenyl)-1H-pyrrol-2-yl)-3-methylbenzamide; 4-(1-(6-hydroxypyridin-3-yl)-3-(4-methoxyphenyl)-1H-pyrrol-2-yl)-3-methylbenzamide; 4-(2-(4-carbamoyl-2-methylphenyl)-3-(4-methoxyphenyl)-1H-pyrrol-1-yl)benzoic acid; 4-(2-(4-carbamoyl-2-methylphenyl)-3-(4-methoxyphenyl)-1H-pyrrol-1-yl)-2-chlorobenzoic acid; 4-(3-(4-methoxyphenyl)-1-(2-(trifluoromethylsulfonamido)ethyl)-1H-pyrrol-2-yl)-3-methylbenzamide; 4-(3-(4-methoxyphenyl)-1-(4-sulfamoylphenyl)-1H-pyrrol-2-yl)-3-methylbenzamide; 4-(1-(3-fluoro-4-hydroxyphenyl)-3-(4-methoxyphenyl)-1H-pyrrol-2-yl)-3-methylbenzamide; 5-(2-(4-carbamoyl-2-methylphenyl)-3-(4-methoxyphenyl)-1H-pyrrol-1-yl)picolinic acid; 4-(1-(3,5-difluoro-4-hydroxyphenyl)-3-(4-methoxyphenyl)-1H-pyrrol-2-yl)-3-methylbenzamide; 4-(1-(5-hydroxypyridin-2-yl)-3-(4-methoxyphenyl)-1H-pyrrol-2-yl)-3-methylbenzamide; 5-(2-(4-carbamoyl-2-methylphenyl)-3-(4-methoxyphenyl)-1H-pyrrol-1-yl)-2-hydroxybenzoic acid; 6-(2-(4-carbamoyl-2-methylphenyl)-3-(4-methoxyphenyl)-1H-pyrrol-1-yl)nicotinic acid; 4-(1-(3-fluoro-4-hydroxyphenyl)-3-(4-(2-methyl-1H-imidazol-1-yl)phenyl)-1H-pyrrol-2-yl)-3-methylbenzamide; and 4-(1-(3,5-difluoro-4-hydroxyphenyl)-3-(4-(2-methyl-1H-imidazol-1-yl)phenyl)-1H-pyrrol-2-yl)-3-methylbenzamide.
 6. The method of claim 1 comprising a pharmaceutical composition comprising a therapeutically effective amount of a compound according to claim 1 together with a pharmaceutically accepted carrier or excipient.
 7. The method of claim 1, wherein said chronic inflammatory disease is selected from the group consisting of inflammatory bowel disease (IBD), Crohn's disease, colitis, psoriasis, and AIDS related dementia.
 8. The method of claim 7, wherein said chronic inflammatory disease is inflammatory bowel disease (IBD).
 9. The method of claim 7, wherein said chronic inflammatory disease is Crohn's disease.
 10. The method of claim 7, wherein said chronic inflammatory disease is colitis.
 11. A method of making a pharmaceutical composition comprising the step of combining a compound of formula I:

wherein R₁ is selected from the group consisting of R₅ and R₆; R₁′ is R₅ when R₁ is R₆, and R₆ when R₁ is R₅; R₂ is selected from the group consisting of hydrogen, halogen, hydroxyl, C₁-C₆ alkyl, C₃-C₆ cycloalkyl, cyano, nitro, trifluoromethyl, carbamoyl, C₁-C₆ alkylcarbamoyl, amino, C₁-C₆ alkylamino, C₁-C₆ dialkylamino, C₁-C₆ alkoxyl, and C₃-C₆ cycloalkoxyl; R₃ is selected from the group consisting of halogen, hydroxyl, carbamoyl, substituted carbamoyl, C₁-C₆ alkylcarbamoyl, sulfamoyl, C₁-C₆ alkylsulfamoyl, C₁-C₆ alkyl, C₃-C₆ cycloalkyl, cyano, nitro, amino, CF₃, carboxyl, ureido, sulfamoylamino, 2-amino-2-oxoethyl, C₁-C₆ alkylamino, C₁-C₆ dialkylamino, arylamino, heteroarylamino, C₁-C₆ alkoxyl, C₃-C₆ cycloalkoxyl, aryl, substituted aryl, heteroaryl, and substituted heteroaryl; R₄ is selected from the group consisting of hydrogen, hydroxyl, halogen, C₁-C₆ alkyl, C₃-C₆ cycloalkyl, cyano, nitro, carbamoyl, C₁-C₆ alkylcarbamoyl, sulfamoyl, C₁-C₆ alkyl sulfamoyl, amino, C₁-C₆ alkylamino, C₁-C₆ dialkylamino, C₁-C₆ alkoxyl, and C₃-C₆ cycloalkoxyl; R₅ is selected from the group consisting of aryl, substituted aryl, heteroaryl, and substituted heteroaryl; R₆ is selected from the group consisting of —CR₇R₈CR₉R₁₀(CR₁₁R₁₂)_(n)R₁₃ and

R₇, R₈, R₉, R₁₀, R₁₁, and R₁₂ are each independently selected from the group consisting of hydrogen, hydroxyl, halogen, C₁-C₆ alkyl, C₃-C₆ cycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl and wherein R₉ and R₁₀ together form a C₃-C₆ cycloalkyl; R₁₃ is selected from the group consisting of COOH and NH₂SO₂CF₃; R₁₄ is selected from the group consisting of OH, COOH, and SO₂NH₂; R₁₅ and R₁₆ are independently selected from the group consisting of hydrogen, hydroxyl, carboxyl, NO₂, and halogen; n is 0 or 1; and X and Y are independently selected from the group consisting of C and N; with a pharmaceutically accepted carrier or excipient. 